Ann Arvin - US grants

Because there are vaccines for other viral diseases, varicella has emerged as the childhood exanthem with the highest risk of serious complications. As antiviral drugs and varicella vaccines are developed, decisions must be made about their administration to normal children, which requires a better understanding of varicella-zoster virus (VZV) immunity in the normal host. A sensitive radioimmunoassay for VZV specific IgG, IgM and IgA antibodies will be used to analyze early humoral immumity in children with varicella. Although deficiencies of cellular immunity are associated with severe variacella, information about cellular immunity to VZV is limited. T-lymphocyte proliferation and T-lymphocyte cytotoxicity will be assessed in this study. Studies of other viral infections indicate that T-lymphocyte cytotoxicity is an important early immune response; this response has not been studied in children with varicella. The analysis of immunity to specific VZV proteins following natural varicella is important for the evaluation of varicella-vaccine induced immunity and of the possible development of VZV sub-unit vaccines. VZV monoclonal antibodies will be used to purify VZV proteins for assays of humoral and cellular immunity to components of the virus. These methods will be used to analyze the responses of normal and immunocompromised children with varicella, VZV immune subjects and varicella vaccine recipients. The possibility that laboratory tests can be used to predict the severity of varicella will be investigated. The identification of such laboratory markers for the likelihood of serious infection would be very helpful in decisions about the initiation of antiviral therapy. The feasibility of producing human monoclonal antibodies to VZV has been demonstrated in preliminary studies. Antobodies produced by human hybridomas will be characterized by immunoprecipitation to determine reactivity with VZV proteins and for functional antiviral activity by neutralization. The production of a human VZV monoclonal antibody with potent neutralizing activity could provide an effective preparation for clinical use as passive antibody prophylaxis for varicella in high risk patients. Further studies of T-lymphocyte mediated immunity during acute varicella are planned to expand upon our observation that the expression of HLA antigens is increased on circulating T-lymphotyes from normal subjects with varicella and, particularly, that HLA-DR expression is present. These experiments are intended to take advantage of the unique opportunity for studies of immune response and immunoregulation during a systemic viral infection.

Varicella (chicken pox) is caused by infection with varicella-zoster virus (VZV). Primary infection with VZV is associated with severe infection in immunocompromised children. Varicella has been considered a benign illness in otherwise healthy children. However, with effective vaccines for other childhood viral infections varicella has emerged as a significant cause of serious illness in the general population. Research concerning VZV has been difficult because of the cell-associated nature of VZV replication in vitro and the lack of animal models for the investigation of VZV pathogenesis. The purpose of this study is to evaluate the strain 2 guinea pig as a model of acute primary VZV infection and of the immune response to primary VZV infection. The guinea pig is uniquely susceptible to infection with human VZV. A viremic phase following inoculation with VZV has been demonstrated in the strain 2 guinea pig. The occurrence of viremia suggests that the guinea pig model can be exploited as an analogue of primary human varicella since viremia appears to be fundamental to the pathogenesis of the initial infection with VZV in the human host. The investigation of the guinea pig as a model of VZV immunity is important given recent efforts to prepare a VZV vaccine. This study will investigate immunity to VZV in the strain 2 guinea pig after inoculation with infectious VZV for comparison with immunity to the virus that can be elicited by immunization with VZV proteins. VZV proteins antigens will be prepared by immunoaffinity using murine monoclonal antibodies to specific VZV proteins. Humoral immunity will be measured by solid phase radioimmunoassay for VZV IgG and IgM antibodies, by VZV neutralization assay and by immune transfer (Western blot). Cellular immunity will be assessed by in vitro T-lymphocite proliferation of peripheral blood mononuclear cells stimulated with VZV antigen. Methods to optimize the detection of viremia will be investigated to facilitate the use of the model to evaluate approaches for restricting VZV replication in vivo. Viral culture of peripheral blood mononuclear cells will be done in parallel with an immunofluorescence method which uses monoclonal antibody to detect VZV antigens on these cells. In situ hybridization with radiolabeled cloned fragments of VZV DNA will be evaluated for the identification of the virus in peripheral blood cells. Protection against viremia will be used as an in vivo biologic measure of the efficacy of immunization with VZV proteins antigens, passive antibody to VZV and antiviral drugs.

Varicella-zoster virus (VZV), a human herpesvirus, causes varicella (chickenpox) and herpes zoster (singles). VZV disease produces serious morbidity and can be life-threatening in otherwise healthy adults, the elderly, and in human immunodeficiency virus (HIV)-infected, cancer and transplant patients. Our objective is to define immunogenic proteins and host factors affecting cellular immune responses to VZV in experiments which will be relevant to VZV vaccine design and optimal use of live attenuated varicella vaccine. Helper and cytotoxic T-cell (CTL) recognition of structural/regulatory gene products (ORF4, ORF10, ORF61, and ORF63) will be evaluated using VZV-vaccinia recombinants. Since memory T-cells recognize gp I, gp IV and IE62 protein, immunodominant regions will be identified with recombinants that express truncated forms of each protein. T-cell epitopes will be mapped using peptides that fit proposed overlapping CD4+ and DC8+ T-cell motifs and expressing the sequences in vaccinia. The dendritic cell system will be developed as an in vitro method to determine whether the same VZV proteins or peptides that elicit T-cell recognition after natural infection in vivo are active primary immunogens when presented as purified proteins or related peptides to naive T-cells. Since the expression of viral proteins in infected cells does not predict their immunogenicity, this method has broad potential value for designing protein or peptide vaccines. Genetic factors affecting the host response will be determined by analyzing T- cell recognition of glycoproteins and IE62 protein in donors of known MHC phenotype. Age-related factors influencing the primary host response will be assessed by comparing VZV specific CD4+ and DC8+ responses in children and adults immunized with varicella vaccine. Cytokine production will be investigated to determine whether adults have a relative failure to prime Th1-like CD4+ T-cells or a relative predominance of the Th3-like subset which could downregulate essential Th1-like responses. Whether the increased susceptibility to VZV reactivation in elderly individuals correlates with a quantitative decrease in T-cells that recognize VZV proteins and/or in the relative predominance of Th1 or Th2-like CD4+ T-cell responses will be evaluated. Understanding cytokine responses to viral antigens could yield new insights about their potential value as vaccine components. Finally, because the widespread use of varicella vaccine will reduce opportunities to boost VZV immunity by re-exposure during the annual varicella epidemics, it is important to investigate whether there ar any age- related obstacles to using the vaccine to enhance T-cell immunity. Helper and CTL recall responses will be evaluated in children and adults with vaccine-induced immunity who ar re-vaccinated after 4-10 years. The comparative efficacy of immunization for enhancing natural immunity will be assessed in younger and elderly adults since VZV vaccines may have therapeutic value for preventing herpes zoster. Investigating VZV immunity is directly relevant to clinical practice because current evidence is that a single vaccine preparation will be effective optimally in all clinical circumstances.

Since antepartum cultures for asymptomatic shedding of HDV from women with elicitable histories of recurrent genital HSV infections do not predict shedding of HSV at delivery, and since up to 77% of mothers of infants infected with HSV do not have an elicitable history of recurrent genital infections, it is proposed to change tactics, identify a high risk population antepartum, and screen for shedding of HSV at delivery. Specific infants exposed to HSV could then be evaluated and, when appropriate, treated. Detection of shedding at delivery is complicated by the fact that HSV has been isolated from the infant only as frequently as from the mother. Amniotic fluid entrapped in the infant's mouth may be best sample and one obtainable in large quantity as compared to swab cultures. Asymptomatic shedding occurs at least as frequently from the lesion site as from the cervix in women with overt genital recurrences; the same may be true in women who are unaware of having genital HSV infections. Since HSV causes 98% of recurrent genital infections in the U.S., it is proposed that women with antibody to HSV 2 be selected as a high risk group using a serological ELISA assay augmented by the use of an avidin-biotin-perioxidase complex. The rate of asymptomatic shedding in those who never perceive overt recurrences will be compared with those who have overt recurrences, those with HSV 1 antibody only, and women with partners with genital HSV infections. The best site(s) to examine for asymptomatic shedding will be assessed by otaining multiple cultures from the mother and infant. Asymptomatic shedding or initial infection during pregnancy will be studied in asymptomatic women with partners with overt genital recurrences and correlated with the woman's part immunological experience with HSV 1 and 2. The ongoing effort to assess the source of HSV infection in infected infants will continue. In view of our belief that the high prevalence of serious HSV infection in the first month of life is related to responses in host resistance rather than solely maternal exposure, and to determine why premature infants are disproportionately represented among infected infants, we plan a re-evaluation of methodology which might prove useful in quantitating early host defenses (quality of passively acquired antibody (HSV 1 vs HSV 2; to glycoproteins vs other proteins) and macrophage/monocyte function). Using an ELISA blot from SDS-PAG gels to assess antibody, a neutralization assay, production and responses of monocytes to an HSV induced chemotactic agent, neutralization and lymphocyte transformation responses, early vs late immune responses will also be studied in treated infants.

Animal models of VZV infection are needed for studies of VZV vaccine strategies and to test newer antiviral agents against VZV. We propose to continue to investigate VZV infection in the strain 2 guinea pig model of VZV because of its value for the analysis of VZV immunity and its potential for studies using viremia as a biological marker in primary VZV infection. Our experiments will focus upon immunomodulation to enhance the response to two major VZV proteins, the glycoprotein I, and p 170, a non- glycosylated VZV phosphoprotein, in the strain 2 guinea pig. Methods of incorporating these proteins into polymeric adjuvants, biodegradable microspherules or liposomes to facilitate antigen presentation in vivo will also be evaluated. The guinea pig VZV system will be most valuable if biological markers can be identified which will correlate with the reduction of viral replication in animals that have been immunized before viral challenge or treated with antiviral agents during the acute phase of the infection. Our strategy has been to use VZV viremia as a marker of particular relevance in VZV pathogenesis since viremia is a critical event during primary VZV infection of human subjects. A VZV DNA probe consisting of the HindIII a, b, c, and d fragments has been developed to identify VZV in peripheral blood cells by in situ hybridization. The VZV DNA probe will also provide an important tool for analyzing issues in VZV pathogenesis that can be addressed only in the animal model, e.g., the investigation of VZV infection of nasopharyngeal mucosal cells. Our preliminary results indicate that VZV viremia can be potentiated by selective immunosuppression, which is analogous to the observation of persistent viremia associated with progressive disseminated varicella infection in immunocompromised children. Other likely target organs of primary VZV infection in the strain 2 guinea pig, e.g., lungs spleen and liver, will be examined for evidence of VZV infection particularly in animals in which VZV viremia has been potentiated by immunosuppressive regimens. Viral culture of tissue samples will be done by cocultivation with guinea pig embryo fibroblasts and tissue sections will be examined by immunofluorescence or immunoperoxidase staining with VZV monoclonal antibodies and by in situ hybridization with the VZV DNA probe. A new rationale for further work with VZV in the guinea pig system is the potential for the study of VZV pathogenesis using viral strains that have been modified by recombinant DNA techniques. Recombinant strains of VZV which express the E. coli lacZ gene will be developed; cells infected by these VZV recombinants in strain 2 guinea pigs will be identified by staining with a chromogenic substrate for beta-galactosidase. The guinea pig model of VZV infection as developed by using in situ hybridization to detect viremia, by immunosuppressive regimens and by the use of genetically altered strains of VZV will provide the basis for experiments to determine the efficacy of VZV immunization strategies and of antiviral agents that inhibit VZV.

The incidence of neonatal HSV infections is increasing in parallel with the increase in genital HSV infections. Preventing most cases of neonatal HSV infections depends upon avoiding contact with the virus at delivery. If lesions are present, delivery by cesarean section is indicated. Unfortunately, most neonatal infections result from exposure to asymptomatic maternal excretion of HSV, often by women with no past history of genital herpes. The failure of antepartum cultures to predict asymptomatic shedding at delivery in women with past recurrent genital herpes along with the fact that many mothers of infants with neonatal HSV have no history of genital herpes requires another approach to the problem of neonatal HSV. Most genital HSV infections are caused by HSV-2. Seroepidemiologic studies have been hampered by cross-reactivity between HSV-1 and 2 antigens until the development of serologic methods which detect antibodies to HSV-2 specific glycoproteins. We propose to investigate the value of taking viral cultures for HSV at all deliveries, regardless of maternal herpes history, and of HSV-2 specific serologic testing early and late in gestation. Past or recent HSV-2 infection will be determined by testing paired sera from the first prenatal visit and 28-32 weeks gestation using an ELISA method to detect antibodies to the HSV-2 glycoprotein G, which has HSV-2 specific epitopes. The frequency of HSV-2 infections in consecutive pregnant women with or without a history of genital HSV and the proportion which are primary infections will be assessed. Cultures will be obtained from all mothers and infants at delivery (approximately 5000/year). The sensitivity and specificity of shell vial culture and HSV antigen detection by enzyme immunofiltration for rapid diagnosis of neonatal HSV exposure will be compared with a standard tissue culture technique. The frequency of asymptomatic shedding of HSV at delivery among women with or without serologic evidence of past or recent HSV-2 infections will be determined. The frequency of prematurity, low birth weight, and/or evidence of intrauterine HSV infections among neonates born to mothers with serologic evidence of HSV-2 infections will be assessed. Much of the continued morbidity and mortality of neonatal HSV is due to delayed diagnosis. While delivery cultures will not prevent the exposure of infants to asymptomatic maternal HSV, identification of exposed infants should allow early diagnosis and immediate antiviral therapy of neonatal HSV. Infants known to be exposed to asymptomatic maternal HSV will be monitored to determine the frequency of subclinical and clinical HSV infections. Exposed neonates who do not contract HSV will be compared with HSV- infected neonates referred during the study period using assays for HSV neutralizing antibody, antibody mediating cellular cytotoxicity and antibodies to HSV glycoproteins. The failure of antepartum cultures to predict and therefore prevent neonatal exposures to HSV at delivery has made it critical to develop a rational approach to the problem of neonatal HSV infections.

The incidence of HSV infection is one in 2,500-5,000 live births, with a higher incidence in certain areas of the United States. Approximately 45-50% of those infected have disease limited to skin, eye, and mouth. Unfortunately, 10% of these babies still have significant neurological deficits. Part I: The purpose of this study is to determine if high dose acyclovir (60 mg/kg/day) is safe and well tolerated in the newborn and whether it appears to decrease morbidity and mortality of central nervous system (CNS) or disseminated neonatal herpes simplex virus (HSV) infection both acutely and on long term follow-up. Part II: The purpose of this study is to determine if suppressive therapy with oral acyclovir will decrease the recurrence of skin lesions in neonatal herpes simplex viurs (HSV) infections involving skin, eye and mouth, and eliminate the long term neurologic impairment. In earlier studies it was noted that the number of skin recurrences correlated directly with the degree of neurological impairment, even with initial parenteral therapy. This would imply a possibility of reactivation of the virus at other sites over time. Suppressive therapy may have the potential to prevent this reactivation and thus eliminate neurological impairment in this group.

Age-related differences in the persistence of immunity to varicella- zoster virus (VZV) were evaluated in 17 adults and 62 children previously immunized with various doses of live attenuated varicella vaccine. Immune response was evaluated by ELISA and VZV- specific lymphocyte stimulation assay. The presence of TH1 and Th2 type cytokines released by T lymphocytes stimulated with VZV was also evaluated. At one year post-vaccination, antibody seropositivity rates were 100% for adults and 94% for children; at five years post- vaccination seropositivity rates were 94% of adults and 95% of children. Cell mediated immunity to VZV was maintained in 94% of adults and 81% of children at five years post-vaccination. Mean stimulation indices (SI) were significantly higher (p=0.04, Student's t test) at the persistence bleed compared to one-year bleed in both adults and children. Cytokine responses (IL-2, IL-10 and INF-y) to VZV antigen were equivalent in adults and children. In conclusion, at five years post-vaccination, no differences in the persistence of immunity to VZV were observed in adults versus children as measured by ELISA, VZV-specific lymphocyte stimulation assay and citokine production.

Age-related differences in the persistence of immunity to varicella- zoster virus (VZV) were evaluated in 17 adults and 62 children previously immunized with various doses of live atenuated varicella vaccine. Immune response was evaluated by ELISA and VZV-specific lymphocyte stimulation assay. The presence of Th1 and Th2 type cytokines released by T lymphocytes stimulated with VZV was also evaluated. A one year post-vaccination, antibody seropositivity rates were 100% for adults and 94% for children; at five years post- vaccination seropositivity rates were 94% of adults and 95% of children. Cell-mediated immunity to VZV was maintained in 94% of adults and 81% of children at five years post-vaccination. Mean stimulation indices (SI) were significantly higher (p=0.04, Student's t test) at the persistence bleed compared to the one-year bleed in both adults and children. Cytokine responses (IL-2, IL-10 and INF-y) to VZV antigen were equivalent in adults and children. In conclusion, at five years post- vaccination, no differences in the persistence of immunity to VZV were observed in adults versus children as measured by the ELISA, VZV- specific lymphocyte stimulation assay and cytokine production.

Reactivation of varicella-zoster virus (VZV) causes herpes zoster, which is common after bone marrow transplantation and is associated with considerable morbidity and even with life-threatening infection in some patients. The goals are to investigate the immunologic correlates of protection against VZV reactivation after hematopoietic cell transplantation (HCT), the role of viral tropism for human T cells in the pathogenesis of recurrent VZV infections, and the contributions of two novel VZV glycoproteins, gM and gN, to infectivity for T cells and skin. We propose to continue our studies of the reconstitution of VZV immunity by administration of inactivated varicella vaccine in a placebo controlled trial that incorporates a focus on refined analysis of the possible mechanisms and immunologic correlates of protection. The vaccine preparation to be tested will also be of higher initial virus content. The evidence from our current studies is that the inactivated vaccine accelerates reconstitution of VZV specific CD4 T cells in autologous HCT recipients who are vaccinated before as well as after transplant. We will continue to evaluate VZV immune reconstitution using novel assays to quantitate the CD8 as well as CD4 T cell responses in vaccine recipients. Immunologic correlates of protection will be defined by prospective monitoring for VZV-specific memory T cell recovery, and for the occurrence of herpes zoster as well as subclinical VZV reactivations documented by real time polymerase chain reaction (PCR). VZV infects human CD4 and CD8 T cells, which allows transport of the virus to visceral organs; this lymphotropism is an important event in the pathogenesis of VZV infections after HCT since it is responsible for the most serious complications of herpes zoster, such as pneumonia and encephalitis. In viral pathogenesis experiments, we plan to continue our focus on the contributions of VZV glycoproteins. The objective is to use our cosmid approach to generate recombinant VZV strains with modifications or deletions of the newly identified VZV glycoprotein genes, gM and gN. These genes are likely to be dispensable in vitro, but may be manipulated to reduce VZV virulence in vivo, providing a new approach for VZV attenuation. VZV recombinants that have deletions or selected mutations in gM and gN will be evaluated for changes in skin and T cell tropism in vivo in the SCID-hu model. A comprehensive assessment of immunologic correlates of protection from herpes zoster after immunization with inactivated varicella vaccine should suggest ways to enhance the control of VZV reactivation after HCT through targeting restoration of particular host responses; this work should have direct relevance to strategies for optimal reconstitution of host responses to other viral pathogens in high risk patients who have impaired immune function. Better understanding of VZV tropism for T cells and skin, and the contributions of the viral glycoprotein genes to virulence will provide basic information that may allow the design of live attenuated VZV vaccines which are safe and immunogenic in immunocompromised patients.

Age-related differences in the persistence of immunity to varicella- zoster virus (VZV) were evaluated in 17 adults and 62 children previously immunized with various doses of live attenuated varicella vaccine. Immune response was evaluated by ELISA and VZV- specific lymphocyte stimulation assay. The presence of Th1 and Th2 type cytokines released by T lymphocytes stimulated with VZV was also evaluated. At one year post-vaccination, antibody seropositivity rates were 100% for adults and 94% for children; at five years post- vaccination seropositivity rates were 94% of adults and 95% of children. Cell-mediated immunity to VZV was maintained in 94% of adults and 81% of children at five years post-vaccination. Mean stimulation indices (SI) were significantly higher (p=0.04, Student's t test) at the persistence bleed compared to one-year bleed in both adults and children. Cytokine responses (IL-2, IL-10 and INF-y) to VZV antigen were equivalent in adults and children. In conclusion, at five years post-vaccination, no differences in the persistence of immunity to VZV were observed in adults versus children as measured by ELISA, VZV-specific lymphocyte stimulation assay and cytokine production.

Age-related differences in the persistence of immunity to varicella- zoster virus (VZV) were evaluated in 17 adults and 62 children previously immunized with various doses of live attenuated varicella vaccine. Immune response was evaluated by ELISA and VZV-specific lymphocyte stimulation assay. The presence of Th1 and Th2 type cytokines released by T lymphocytes stimulated with VZV was also evaluated. At one year post-vaccination, antibody seropositivity rates were 100% for adults and 94% for children; at five year post- vaccination seropositivity rates were 94% of adults and 95% of children. Cell-mediated immunity to VZV was maintained in 95% of adults and 81% of children at five years post-vaccination. Mean stimulation indices (SI) were significantly higher (p=0.04), Student's t test) at the persistence bleed compared to the one-year bleed in both adults and children. Cytokine responses (IL-2, IL-10 amd INF-y) to VZV antigen were equivalent in adults and children. In conclusion, at five years post- vaccination, no differences in the persistence of immunity to VZV were observed in adults versus children as measured by Elisa, VZV-specific lymphocyte stimulation assay and cytokine production.

Varicella-zoster virus (VZV) causes varicella and herpes zoster. Our goal is to improve knowledge about how this common pathogen causes disease and about protection provided by natural and vaccine-induced immunity. Glycoproteins are likely to be host range determinants for T cells and skin, which are critical target cells during VZV infection. Our focus is on glycoproteins, gI (ORF67) and gE (ORF68). The effect of gI or gE mutations made in cosmids, on VZV replication will be determined in vitro. Infectivity for human CD4+ and CD8+ T cells or skin will be assessed in vivo in the SCIDhu mouse model of VZV pathogenesis, which reveals critical roles for VZV proteins that are completely dispensable in tissue culture. T cell tropism will also be investigated in thymic organ cultures and II23 cells, a CD4+ T cell hybridoma, using green fluorescent protein (gfp)-labeled VZV. VZV gI and gE effects on epithelial cells will be evaluated in MDCK cells. The vaccine strain, V-Oka, will be compared with its parent, P-Oka, to determine whether gE or gI mutations explain V-Oka attenuation. VZV infects T cells in the naive host and spreads before VZV specific immunity is induced. We have found that VZV interferes with cell surface expression of major histocompatibility (MHC) class I and class II. Our goals are to identify viral immunomodulatory proteins that allow VZV to escape from immune surveillance and to determine whether skin homing receptors facilitate transport of infected T cells to skin. Whether these mechanisms function at skin sites during natural infection will be determined in biopsies from acute varicella lesions. Rapid acquisition of VZV specific T cell responses correlates with mild varicella and maintenance of latency. We propose to address important questions about adaptive VZV immunity with new methods to measure CD4+ and CD8+ T cell responder frequencies against dominant viral proteins, gE and the immediate early tegument/transactivating protein, IE62. We will examine differences in protection afforded by natural and vaccine-induced immunity, diminished immunogenicity of varicella vaccine in adults, and declining VZV T cell responses with aging. Quantitative comparisons of CD4+ and CD8+ recognition of gE and IE62 protein and peptides will be made using intracellular cytokine assays. Peptides appropriate for synthesis as MHC class I and class II tetramers will be identified and used to enumerate VZV specific responder T cells in CD4+ and CD8+ subsets. These parallel investigations of VZV pathogenesis and immunity are directly linked by their practical relevance for improving live attenuated varicella vaccines.

The rate of acquisition of human cytomegalovirus (HCMV) infection is very high during the first two years of life. Most infections are asymptomatic in young children, but the virus can be transmitted to susceptible pregnant women, causing severe neurologic damage to the fetus. Very little is known about virus-specific CD8 T cell responses in young children. HCMV infection provides an excellent context in which to examine the functional maturation of this component of antiviral immunity in healthy children because virus excretion in urine can be quantitated and monitored in parallel with immunologic studies. We propose to examine whether HCMV-specific CD8 T cells contribute to resolution of active primary HCMV infection in early childhood. Highly sensitive flow cytometry techniques requiring small amounts of peripheral blood afford an unprecedented opportunity to assess the capacity of the developing immune system to respond to this systemic viral infection. CD8 T cell responses will be correlated with changes in viral 'load', measured by virus titration of urine. Intracellular cytokine (ICC) assays will be carried out using HCMV-infected cell lysate and pp65 protein as antigens, with CD69 as the marker of T cell activation and IFN-gamma as the marker of the antigen-specific response. HLA-A2 positive infants will also be tested for acquisition for HCMV-specific CD8 T cells using a class I-HCMV pp65/495-503-A2 tetramer. In order to evaluate the prevalence of 'effector' versus 'memory' phenotypes and the functional characteristics of HCMV-specific CD8 T cells, HCMV- specific CD8 T cells that are detected in infected infants will be characterized for cell surface phenotypes using antibodies to RA and CD27. These experiments will determine whether 'effector' or 'memory' CD8 T cell subpopulations predominate at early or late times during the course of primary HCMV infection. The same infants will be tested for HCMV-specific CD4 T cell responses in Project 1 while viral tropism for peripheral blood cells and maturation of antigen presentation will be studied in Project 3. Defining whether the HCMV-specific CD8 T cell response is a correlate of reduce viral replication in the healthy host is important to HCMV vaccine design, providing criteria for immunogenicity based upon similarities to natural immunity. More generally, investigation of CD8 T cell responses to HCMV antigens will help to elucidate the contribution of this arm of the immune system to the control of viral infections in early childhood. This information is relevant for the design of vaccines against other viral disease acquired in infancy of early childhood.

The fundamental objective of this Vaccine Immunology Basic Research Center is to address the deficiencies in knowledge about the developing immune system. This proposal on 'Antiviral Immune Mechanisms in Early Childhood' will enhance knowledge about the developmental biology of the host response in infants and young children, emphasizing adaptive virus-specific immunity mediated by CD4 and CD8 T cells, and the postnatal ontogeny children, emphasizing adaptive virus-specific immunity mediated by CD4 and CD8 T cells, and the postnatal ontogeny of antigen-presenting cell function. Whereas most viral pathogens are encountered during the first few years of life, and most viral vaccines must be given during this interval in order to provide maximum benefit, very little is known about the acquisition of memory T cell immunity in the young child. Human cytomegalovirus (HCMV) is a 'model' since it is common in early childhood, HCMV is readily detected in urine, and immunologic control of infection can be defined in relation to cessation of virus shedding. Three Research Projects will use innovative flow- cytometry based methods to analyze antiviral immune mechanisms in young children, including Project 1: Postnatal ontogeny of HCMV- specific CD4 T cell immunity, Project: The evolution of CD8 T cell responses to HCMV in infants, and Project 3: Antigen presentation: ontogeny and modulation by HCMV. Conceptually, these three projects taken as a whole, will define the contributions and interactions between the three components of the adaptive immune response, including CD4 T cells, CD8 T cells and antigen-presenting cells (APC), necessary to achieve effective antiviral immunity. The common focus is of identifying differences between the functional capacities of CD4 T cells, CD8 T cells and APCs in young children compared to adults, that may account for the prolonged interval required for the immature host to control HCMV replication. The goal is to reveal patterns of impaired or delayed immunity in early childhood. The Program Project structure includes a Clinical Research Core, to enroll and monitor subjects, maintain a centralized data base, and provide biostatistical oversight. Characterizing basic immune mechanisms associated with host containment of HCMV has direct practical significance for designing an effective HCMV vaccine, which was classified as highest priority by the 1999 Institute of Medicine, report on vaccines. More generally, improving our knowledge about the maturation of adaptive immune responses in early childhood will be used for the many major initiatives now aimed at developing new viral vaccines and improving the safety and efficacy of existing vaccines for use in young children.

Group A Rotaviruses are highly important human pathogens. Estimates of their impact on man include worldwide mortality rates of 800,000 to 1 million infant deaths annually. Despite substantial progress over the past two decades in determining rotavirus gene structure and function, we still do not have a clear understanding of the basis of rotavirus immunity, virulence or host tropism. It is the purpose of this proposal to continue our investigation of several aspects of rotavirus pathogenesis, including host range restriction, virulence and immunity. The specific aims of this application are: 1) To identify the viral and immunologic determinants of protective immunity in vivo. We will carry out a series of experiments in a mouse model to correlate the quantity and specificity of the local and systemic humoral immune response with protection. In order to determine why homologous infection induces local IgA responses more efficiently than heterologous infection, we will characterize the T helper cell response in the gut and spleen after murine and non-murine rotavirus infection of suckling mice. We further propose to identify and characterize the viral targets of protective immunity. Protection studies will be carried out in the adult mouse challenge model with homologous, heterologous and ressortant viruses that vary serologically. Immunization studies will also be carried out using specific recombinant viral gene products or killed virion delivered to various mucosal or systemic sites. We plan to determine which specific arm of the immune system mediates protective immunity. Passive transfer studies will be carried out employing isolated populations of immune effectors cells (CD4+, and/or CD8+T cells and/or B cells derived from various tissues) from immune MHC-matched donors. We will also study the cellular determinants of protective immunity using mice deficient in specific components of the immune system. 2) To identify the viral and immunologic determinants for the resolution of rotavirus infection. We will determine if CD4+ as well as CD8+ T cells or local IgA antibody are capable of mediating resolution of infection by themselves when passively transferred into chronically infected SCID mice. Mice deficient in specific immune functions will be also be examined to determine if they ar fully or partially deficient in their ability to resolve infection and/or if immune cells taken from such animals can eliminate rotavirus infection in chronically infected SCID mice. 3) To determine the target of and mechanism by which IgA and other monoclonal antirotavirus antibodies prevent, and possibly resolve, rotarvirus infection in vitro and in vivo. In order to study the role of IgA directly we will carry out challenge studies in mice bearing transplanted rotavirus-specific IgA secreting hybridomas. Finally, we will investigate the mechanism by which IgA and IgG neutralizing monoclonals directed at VP7 or VP4 inhibit viral replication. 4) To determine the genetic and molecular basis of host range restriction and virulence. It is our hypothesis that the surface proteins of rotavirus are important but not exclusive determinants of virulence; that virulence and host range determinants vary depending on the genetic and physiological context of the analysis and that genes encoding non- structural as well as structural proteins may be important determinants of host range restriction. In order to determine the genetic basic of host range restrictions and virulence, we will carry out genotypic and phenotypic analyses of rotavirus reassortants in the infant mouse model. In order to better understand the molecular and virologic basis of virulence and/or host tropism, we will identify relevant steps in the viral replication cycle in vivo that are restricted on the basis of host range.

DESCRIPTION (provided by applicant) Rotaviruses (RVs) are the most important cause of severe dehydrating diarrhea in children in both developed and less developed countries. It is estimated that RV are responsible for the death of approximately 2000 children daily worldwide principally in developing countries. Studies in animals and in humans indicate that humoral immune mechanisms appear to be the primary determinants of protection from reinfection following wild-type disease or vaccination. Better methods are needed to characterize the qualitative and quantitative nature of the humoral immune response in children from developed and less developed countries. The proposed studies will be done primarily in Colombia- South America as an extension of NIH Grant: (R37 AI21362). Using B cell ELISPOTS and a novel flow cytometry assay we plan to quantify and study the phenotype of rotavirus specific B cells induced after natural rotavirus infection in children and adults in Colombia, and in children after natural rotavirus infection and after administration of a rotavirus vaccine. We will study these lymphocytes for the presence of molecules implicated in lymphocyte homing to the intestinal mucosa and B cell maturation markers that will aide in differentiating effector vs. memory B cells. A practical long-term goal of this project is to find parameters that correlate with protection induced by rotavirus vaccines. Since rotaviruses replicate almost exclusively in the intestinal mucosa, another long-term goal of this project is to gain a better understanding of the molecular determinants of the immune response to rotavirus in particular, as well as a deeper understanding of the humoral mucosal immune response in general with specific emphasis on B cell memory and homing.

This Cooperative Center for Translational Research on Human Immunology and Biodefense is entitled 'Influenza Immunity: Protective Mechanisms against a Pandemic Respiratory Virus'. Our objective is to use vaccine-induced and naturally acquired influenza A immunity as a model for comprehensive, integrated analyses of adaptive and innate immune mechanisms and antimicrobial protection of the respiratory tract in children and adults. Influenza immunology is relevant to biodefense because influenza A has significant potential to be modified genetically to create a bioterrorist agent. Further, influenza A causes natural pandemics, which can incapacitate a large fraction of the population, endangering preparedness. Influenza A has many characteristics of microbial pathogens that could become agents of civilian bioterrorism. Among these are: capacity to cause illness with high morbidity and mortality, highly efficient person-to-person transmission, high infectivity by aerosol, resulting in the capacity to cause large outbreaks, potential to cause anxiety in the public, and potential to be weaponized. While influenza vaccines exist, the immunologic mechanisms by which protection is induced in the respiratory tact are poorly understood in the human host. Genetically altered influenza A viruses that express unique hemagglutinin (HA) and neuraminidase (NA) proteins have the capacity to infect all age groups. In a biodefense context, the rapidity with which protection can be elicited in a non-immune population is critical. The influenza A model is expected to allow a better definition of specialized adaptive B cell and T cell immune mechanisms that control infections of the respiratory system. Our investigative approach also encompasses the study of innate, natural killer cell responses to influenza, in parallel with acquisition of adaptive immunity in children and adults. Comparing influenza vaccines will identify differences when the host responds to parenterally administered, inactivated antigens, versus live attenuated virus delivered via the respiratory route. At our Center, investigators leading the Research Resource Technical Development component and the Research Projects will undertake rapid translation of basic immunology methods into applications for analyzing innate and acquired influenza A immunity. These innovations will have broad relevance for understanding human immunity against microbial pathogens of concern for biodefense.

vertical transmission; low birth weight infant human; cytomegalovirus; Herpesviridae disease; breast feeding; premature infant human; longitudinal human study; human milk; clinical research; human subject;

DESCRIPTION (provided by applicant): Varicella zoster virus (VZV), a human alphaherpesvirus, causes varicella and herpes zoster, and has a significant impact on public health. We will pursue new opportunities to understand molecular mechanisms of VZV pathogenesis using VZV cosmids to construct VZV mutants and SCID mouse models with skin, T cell and dorsal root ganglia (DRG) xenografts. Varicella vaccines (vOka) are effective in healthy children but retain capacities to cause viremia and latency. Our goal is to use VZV cosmids and the SCIDhu skin, T cell and neural models to define options for an improved varicella vaccine. We will investigate 1) roles of glycoproteins, gE and gl in pathogenesis, and 2) VZV infection of T cells and innate host cell responses as critical interactions preceding adaptive immunity. VZV gE and gl are predicted to be multi-functional proteins critical for cell-cell spread, envelopment and possibly entry. Consequences of specific mutations in gE or gl for growth, gE and gl trafficking, virion envelopment and entry will be determined. rOka gE and gl mutants will be tested to establish functions of gE and gl required for infection of skin, T cells or DRG in vivo. These experiments provide the opportunity to show how gE and gl determine VZV virulence in human neurons, as well as skin and T cells in vivo. We will also assess whether cellular transactivators that influence gE expression are cell-type specific determinants of VZV virulence. The SCIDhu skin model will be used to address questions about humoral immunity, determining if anti-glycoprotein antibodies affect VZV cell-cell spread in vivo or interfere with transfer from T cells to skin. In Aim 2, our model is that T cells transport virus to skin where VZV must overcome innate cellular barriers, enhanced by natural killer (NK) cells. We will examine interferon (IFN-alpha) and the expression of the toll-like receptor 9 in SCIDhu mice with VZV-infected skin xenografts and in vitro, to provide new information about innate cellular defenses against VZV. The effect of NK cells, which release IFN-gamma and tumor necrosis factors (TNF), on expression of adhesion molecules, chemoattractants, and VZV replication will be examined in skin xenografts. Finally, whether VZV-infected T cells transfer VZV to DRG as well as skin, will be examined using parent Oka and vaccine Oka viruses. Better understanding of the mechanisms of VZV tropism for T cells, skin and neural cells, and of innate antiviral immunity, will be useful for improving strategies to prevent varicella and zoster in healthy and immunocompromised individuals.

Herpesvirus infections cause serious morbidity and can be fatal after hematopoietic cell transplantation (HCT). The goal of Project 8 is to explore the hypothesis that early reconstitution of innate antiviral immunity acts in concert with adaptive immunity to control these infections. Specific Aim 1 will assess relationships between reconstitution of natural killer (NK) cells and varicella-zoster virus (VZV)- specific and cytomegalovirus (CMV)-specific T cells and VZV and CMV reactivation. Allogeneic HCT patients will be evaluated at 30 and 90 days and 6 and 12 months after HCT using flow cytometry methods to assess NK cell maturity, receptor repertoire and T cell-dependent IFN^y production and for virus-specific CD4 and CDS T cell frequencies. To establish correlations with protection, patients will be monitored for clinical VZV and CMV disease and subclinical infections, detected by PCR testing of peripheral blood mononuclear cells for viremia. Statistical analyses of relationships among measures of immune reconstitution, viral infection and clinical variables will be done. In Specific Aim 2, we will evaluate innate control of VZV infection in dorsal root ganglia (DRG) xenografts in the SCIDhu mouse model. Innate responses will be investigated using immunohistochemistry to assess interferon (IFN) and Nuclear Factor K-B (NFicB) up-regulation and interleukin-15 (IL-15) expression in VZV-infected and uninfected neurons and non-neuronal cells. Modulation of VZV infection by exogenous IFN-a, IFN-y or IL-15 will be determined by infecting DRG with VZV rOkaF62/63RL, which has a firefly luciferase reporter cassette allowing non-invasive assessment of VZV replication. Whether adoptive transfer of NK cells or cytokine-induced killer cells limits VZV replication in DRG will also be investigated. The SCIDhu DRG model offers a unique opportunity to assess the antiviral effects of cytokines and cellular immunotherapy on VZV replication in sensory ganglia in vivo and should demonstrate whether innate responses can restrict VZV replication in a system that mimics allogeneic HCT. Profiling innate and adaptive immune responses in HCT patients along with surveillance for VZV and CMV reactivation and disease will identify antiviral mechanisms that are important for modifying herpesvirus infections after HCT. Exogenous IFNs and IL-15 are modalities that can be considered as adjunctive therapies in HCT recipients. Adoptive cell therapies, including CIK cells are being evaluated for tumoricidal activity in HCT recipients and could have the incremental benefit of controlling herpesvirus infections. These prospective clinical studies of innate and adaptive immunity to VZV and CMV and experiments in VZVinfected SCIDhu DRG have the potential to yield new insights about antiviral immune mechanisms and to suggest new measures for minimizing the burden of herpesvirus-related disease after HCT.

0-11 years old; 21+ years old; ATGN; Acute; Address; Adhesions; Adult; Age; Age Group Unspecified; Aged 65 and Over; Animal Model; Animal Models and Related Studies; Antigens; Antiviral Agents; Antiviral Drugs; Antivirals; Attenuated; Attenuated Live Virus Vaccine; Attenuated Vaccines; B blood cells; B-Cells; B-Lymphocytes; Blood Sample; Blood specimen; Bursa-Dependent Lymphocytes; Bursa-Equivalent Lymphocyte; CD28; CD28 gene; CD4 Positive T Lymphocytes; CD4 T cells; CD4 lymphocyte; CD4+ T cell; CD4+ T-Lymphocyte; CD4-Positive Lymphocytes; CD56; CD8; CD8B; CD8B1; CD8B1 gene; Cell Function; Cell Process; Cell physiology; Cells, CD4; Cellular Function; Cellular Physiology; Cellular Process; Characteristics; Child; Child Youth; Children (0-21); Clinical Research; Clinical Study; Color; Cytofluorometry, Flow; Data Set; Dataset; Detection; Elderly; Elderly, over 65; Epidemiology / Surveillance; Evaluation; Event; Exposure to; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Flu vaccine; Frequencies (time pattern); Frequency; Grippe; Human; Human, Adult; Human, Child; Human, General; Immune; Immune response; Immunity; Immunization; Immunologic Stimulation; Immunological Stimulation; Immunology; Immunology (Including BRMP); Immunology (NCI Program); Immunostimulation; Individual; Infection; Influenza; Influenza A virus; Influenza Vaccines; Influenza Viruses Type A; Influenza virus vaccine; Investigation; Knowledge; LYT3; Life; Live-attenuated Vaccine; Man (Taxonomy); Man, Modern; Medical Surveillance; Memory; Methods; Methods and Techniques; Methods, Other; Microfluorometry, Flow; NCAM; NCAM1; NCAM1 gene; Nasal; Nose; Nose, Nasal Passages; Numbers; Orthomyxovirus Type A; PCR; Pattern; Phenotype; Play; Polymerase Chain Reaction; Population; Predisposition; Production; R01 Mechanism; R01 Program; RPG; Research Grants; Research Project Grants; Research Projects; Research Projects, R-Series; Respiratory System, Nose, Nasal Passages; Risk; Role; Seasons; Sensitization, Immunologic; Sensitization, Immunological; Subcellular Process; Surveillance; Susceptibility; T memory cell; T-Cells; T-Lymphocyte; T4 Cells; T4 Lymphocytes; T44; Techniques; Testing; Thinking; Thinking, function; Thymus-Dependent Lymphocytes; Time; Translational Research; Translational Research Enterprise; Translational Science; Vaccination; Vaccines; Vaccines, Attenuated; Viral Diseases; Virus; Virus Diseases; Viruses, General; Week; adult human (21+); adult youth; advanced age; age group; base; biodefense; chemokine receptor; children; cohort; elders; experiment; experimental research; experimental study; flow cytophotometry; flu infection; geriatric; helper T cell; host response; human subject; immunogen; immunoresponse; influenza infection; late life; later life; live vaccine; memory T lymphocyte; model organism; older adult; older person; pandemic; pandemic disease; pathogen; peripheral blood; prospective; research study; response; senior citizen; social role; thymus derived lymphocyte; tool; translation research enterprise; viral infection; virus infection; young adult; youngster

Behavior; Biological Function; Biological Process; Cell Function; Cell Process; Cell physiology; Cellular Function; Cellular Physiology; Cellular Process; Classification; Clinical Protocols; DNA Chips; DNA Microarray; DNA Microarray Chip; DNA Microarray format; DNA Microchips; Data; Disease; Disorder; Enrollment; Event; Gene Expression; Genes; Genome; Human; Human, General; Imagery; Immune response; Immunization; Immunologic Stimulation; Immunological Stimulation; Immunostimulation; Individual; Knowledge; Laboratories; Lesion; Life; Man (Taxonomy); Man, Modern; Maps; Markers, Surrogate; Microarray Analysis; Microarray-Based Analysis; Modeling; Monkeys; P. variolae; P.variolae; Pathogenesis; Pattern; Pilot Projects; Poxvirus officinale; Poxvirus variolae; Programs (PT); Programs [Publication Type]; Sensitization, Immunologic; Sensitization, Immunological; Smallpox; Smallpox Viruses; Subcellular Process; Surrogate Markers; Systematics; Vaccinated; Vaccination; Vaccines; Vaccinia; Vaccinia Vaccine; Vaccinia virus; Variola; Variola virus; Viral; Visualization; cDNA Arrays; cDNA Microarray; cohort; design; designing; disease/disorder; emergency service personnel; emergency service responder; emergency service/first responder; enroll; experiment; experimental research; experimental study; first responder; host response; human data; immunoresponse; insight; microarray technology; non-human primate; nonhuman primate; pilot study; programs; recombinant vaccinia virus; research study; response; small pox; small pox virus; variola major

Varicella zoster virus (VZV) causes varicella during primary infection, persists in sensory ganglia and may reactivate from latency to cause zoster. VZV pathogenesis depends upon its tropisms for T cells, skin and sensory ganglia. VZV vaccines to prevent varicella and to reduce zoster morbidity in the elderly are very effective. However, VZV control could be improved with a 2nd generation recombinant vaccine that has attenuated replication in skin, but limited infectivity for T cells and ganglia. VZV infection of sensory ganglia ensures its survival in the human population through reactivations from latency that result in zoster. We have developed a model for studying VZV neuropathogenesis by making xenografts of human dorsal root ganglia (DRG) in mice with severe combined immunodeficiency (SCID). Our analyses of VZV tegument/regulatory proteins will be extended to examine their functions in VZV neurotropism. We will focus on IE63, an important immediate early regulatory protein encoded by ORF63, and the ORF66 kinase protein using VZV recombinants that have targeted mutations in these genes. The SCIDhu DRG model also offers unique opportunities to investigate how cell transactivators that modulate viral gene promoters may control VZV neurotropism and to identify what perturbations of persistently infected neurons may trigger of VZV reactivation. The roles of IE63 and ORF66 at early and late stages of VZV infection of ganglia will be examined in SCIDhu DRG. Experiments will address four general hypotheses: 1) initial VZV replication is required for, or influences the level of persistent VZV DNA copies in neurons;2) VZ virions must be made and released efficiently;3) VZV proteins that inhibit neural cell apoptosis facilitate persistence;4) VZV infection of DRG reflects an equilibrium with innate cellular responses, mediated by interferons (IFN), which optimizes persistence. VZV gene promoters, like those of all herpesviruses, have elements that are recognized by ubiquitous host cell regulatory proteins. Our hypothesis is that cellular proteins regulate transcription from the critical ORF63 promoter during initial infection and are needed for the transition to latency in sensory neurons. Experiments to understand what cellular stressors may induce VZV reactivation from persistently infected DRG will examine heat, chemical agents, histone deacetylase inhibitors, interference with nerve growth factor signaling and other triggers known to enhance herpes simplex virus reactivation. The work proposed for Yr. 1 and Yr. 2 (see Specific Aims) is expected to yield new information about the molecular mechanisms of VZV neuropathogenesis in differentiated peripheral neurons and satellite cells within human sensory ganglia and to identify options for designing a 2nd generation VZV vaccine with genetic changes that have been proved to reduce virulence in the SCIDhu DRG model in vivo.

DESCRIPTION (provided by applicant): Herpes simplex virus-1 (HSV-1) is human alphaherpesvirus that establishes a lifelong latent infection in peripheral nerve ganglia following primary infection. HSV-1 infections are generally benign, although its capacity for neurovirulence and neuroinvasiveness are the primary mechanisms through which HSV-1 can cause harmful disease in humans, especially in neonates and immunocompromised hosts. Our overall objective is to develop a model for examining HSV-1 neuropathogenesis in human sensory ganglia in vivo. We will evaluate HSV-1 infection of human dorsal root ganglion (DRG) xenografts in mice with severe combined immunodeficiency (SCID), exploiting the system that we created to investigate varicella- zoster virus (VZV) neuropathogenesis. The biology of HSV-1 infection is similar to VZV in that both HSV-1 and VZV establish latency within sensory ganglia following primary infection. Studies of VZV in the SCIDhu DRG model have provided the first opportunity to examine replication of a human alphaherpesvirus within cells that comprise human DRG in vivo. The DRG xenograft model has the potential to reveal characteristics of HSV-1 neuropathogenesis in the natural human host tissue microenvironment in vivo in an experimental system that will add substantially to observations from rodent models. Experiments will address three specific aims: (1) we will define the course of events that follows HSV-1 inoculation of human DRG xenografts in SCID mice, identifying what cell types within DRG are permissive for HSV-1 gene expression, whether neurons and/or satellite cells become productively infected and whether HSV-1 undergoes the pattern of transition to persistence in human neurons that we have observed in VZV-infected DRG xenografts;(2) we will investigate HSV-1 gene functions through the evaluation of recombinant HSV-1 strains, in particular we will examine the requirement for HSV-1 thymidine kinase (TK) during initial infection and persistence in DRG, and gD mutants for their capacity for viral entry;(3) if HSV-1 is shown to establish persistence in DRG xenografts, we will assess whether this model can be used to study HSV-1 reactivation by explanting latently-infected DRG xenografts and treating with agents that trigger neural cell signaling pathways and increase HSV-1 reactivation in rodent models. This work is intended to demonstrate the feasibility of using DRG xenografts in SCID mice to explore the molecular mechanisms of HSV-1 neuropathogenesis in differentiated human sensory neurons and non-neuronal cells within their sensory ganglia tissue microenvironment in vivo. In addition to new insights about basic virus-host interactions, such a model has potential value for studying antiviral drugs and live attenuated HSV-1 vaccine candidates to treat or prevent human disease caused by this common virus. PUBLIC HEALTH RELEVANCE: Herpes Simplex Virus 1 (HSV-1) causes oral and genital lesions and encephalitis. These infections remain an important public health problem in the United States. Serious complications from HSV-1 can occur in healthy people and in those who have diseases that impair their immune systems. Our goal is to develop a model to study how HSV-1 infects human nerve cells that will have potential value for developing new drugs and vaccines.

DESCRIPTION (provided by applicant): Varicella-zoster virus (VZV) initiates primary infection by upper respiratory mucosal inoculation, causing varicella. VZV persists in sensory ganglia and reactivation from latency may result in zoster. The VZV genome has at least 71 known or predicted open reading frames (ORFs) but understanding how these gene products function in virulence is difficult because VZV is highly human-specific. We have addressed this obstacle by investigating VZV infection of human skin, T cell and dorsal root ganglia xenografts in the severe combined immunodeficiency (SCID) mouse model. Together with clinical observations, this work supports a new paradigm of VZV pathogenesis that incorporates a critical role for T cells as a vehicle for VZV delivery to both skin and neuronal sites of latency. VZV infects human tonsil T cells very efficiently and infected tonsil T cells transfer VZV into skin xenografts, where lesion formation occurs gradually against a barrier of a potent innate epidermal cell response. VZV-infected T cells also exit from the circulation into DRG xenografts, indicating that VZV can reach neurons by this mechanism. T cell tropism is an important difference between the life cycles of VZV and herpes simplex viruses 1 and 2. We propose state of the art screening methods to show how takeover of T cells by VZV modifies cell protein synthesis and activation and alters T cell gene regulation in combination with experiments to define how specific VZV gene products function to achieve these changes in the infected T cell. We will investigate VZV effects on T cell signaling pathways, T cell protein synthesis and T cell gene expression at single cell level using validated screening techniques that have been applied to studies of human T cell biology;methods will include multiparameter flow cytometric analysis of phosphoproteins, multiplex cytokine/chemokine assays and microfluidics-based profiling of gene expression (Aim 1). Based on our preliminary observations, we will examine the contributions of three VZV proteins, immediate early 4 (IE4), immediate early 62 (IE62) and ORF3 protein to VZV T cell tropism (Aim 2). Screening experiments that identify major VZV-induced consequences for the T cell will set the stage for additional studies of the mechanisms by which the virus achieves these effects. VZV is likely to target some pathways common to other T cell tropic viruses and strategies for applying new technologies to investigate viral pathogenesis should emerge. From a public health perspective, this work should provide information to improve the live attenuated Oka strain vaccines for varicella and zoster. While these vaccines are safe and beneficial, this work will be relevant for developing a 2nd generation VZV vaccine which consists of a VZV recombinant virus that has impaired T cell tropism and therefore less potential to disseminate in high risk patients and to establish latency in healthy individuals. PUBLIC HEALTH RELEVANCE: Chickenpox and herpes zoster (shingles) are caused by varicella-zoster virus (VZV). These infections remain an important public health problem in the United States. The licensed VZV vaccines induce protection in most children and elderly adults but complications can occur in healthy vaccine recipients and in those who have diseases that impair their immune systems. Our goal is to identify strategies for creating better vaccines to prevent VZV infections in healthy and high risk people.

DESCRIPTION (provided by applicant): Varicella-zoster virus (VZV) is a medically important human ¿-herpesvirus that causes varicella (chickenpox) and leads to zoster (shingles) upon reactivation from latently infected sensory ganglia. Varicella can be serious and is life-threatening in immunocompromised patients. VZV exhibits tropism for T cells, skin and neurons during infection of the human host and overcomes the usual constraint against fusion between fully differentiated host cells to form multinucleated polykaryocytes, a hallmark of VZV pathogenesis. Glycoprotein B (gB) along with the gH/gL heterodimer is known to be critical for fusion of the virion envelope with the target cell membrane during herpesvirus entry. Our novel concept is that VZV mediates cell-cell fusion through a gB-dependent intracellular signaling function. This is based on our new evidence that preventing tyrosine phosphorylation of the gB cytoplasmic domain (gBcyt) leads to anomalies in cell-cell fusion and syncytia formation in vitro. Our application will investigate how the gBcyt modulates cell- cell fusion mechanisms via intracellular signaling pathways to produce the characteristic syncytia in vitro and fusion of epidermal cells and neuron-satellite cells caused by VZV infection of skin and ganglia in vivo. In Aim 1 we will determine how VZV modifies cellular regulation to favor transcription of genes that facilitate cell-cell fusion and syncytia formation by applying the high-throughput whole-transcriptome sequencing technology, RNA-seq, to our new fusion assay and our virus mutants, which carry mutations in the gBcyt residues that affect cell-cell fusion. To quantify the effects o tyrosine phosphorylation, the spatiotemporal evolution of syncytia formation will be measured in real-time for VZV and the gBcyt mutants. To determine the role of genes in cell fusion, as identified by RNA-seq, we will perform gene perturbation experiments to assess their biological significance in the context of VZV replication. Aim 2 will determine how the gBcyt regulates intracellular signaling events in cell fusion via post-translational modifications of tyrosine and/r lysine residues by cellular or viral proteins. Mass spectrometry will be used to identify cellular and viral proteins that interact with the gBcyt domain in its tyrosine-phosphorylated and non-phosphorylated forms. Lysine mutagenesis studies will be performed to assess the effects of acetylation and ubiquitination posttranslational modifications on VZV fusion and virulence. Finally, Aim 3 will establish whether the gBcyt modulates polykaryocyte formation to optimize VZV infection of skin and DRG. Our mutant viruses will be compared to wild type VZV for replication competencies in human skin and neuronal tissue using novel reporter viruses. We will establish the role of newly identified genes required for cell-cell fusion using a novel shRNA carrying virus. Given the significance of polykaryocyte formation for pathogenesis, deciphering how VZV regulates this process has the potential to yield new strategies for vaccine virus attenuation and antiviral drug design to ease the burden on vulnerable populations.

Varicella-zoster virus (VZV) initiates primary infection by upper respiratory mucosal inoculation, causing varicella. VZV persists in sensory ganglia and reactivation from latency may result in zoster. The VZV genome has at least 71 known or predicted open reading frames (ORFs) but understanding how these gene products function in virulence is difficult because VZV is highly human-specific. We have addressed this obstacle by investigating VZV infection of human skin, T cell and dorsal root ganglia xenografts in the severe combined immunodeficiency (SCID) mouse model. Together with clinical observations, this work supports a new paradigm of VZV pathogenesis that incorporates a critical role for T cells as a vehicle for VZV delivery to both skin and neuronal sites of latency. VZV infects human tonsil T cells very efficiently and infected tonsil T cells transfer VZV into skin xenografts, where lesion formation occurs gradually against a barrier of a potent innate epidermal cell response. VZV-infected T cells also exit from the circulation into DRG xenografts, indicating that VZV can reach neurons by this mechanism. T cell tropism is an important difference between the life cycles of VZV and herpes simplex viruses 1 and 2. We propose ¿state of the art¿ screening methods to show how takeover of T cells by VZV modifies cell protein synthesis and activation and alters T cell gene regulation in combination with experiments to define how specific VZV gene products function to achieve these changes in the infected T cell. We will investigate VZV effects on T cell signaling pathways, T cell protein synthesis and T cell gene expression at single cell level using validated screening techniques that have been applied to studies of human T cell biology; methods will include multiparameter flow cytometric analysis of phosphoproteins, multiplex cytokine/chemokine assays and microfluidics-based profiling of gene expression (Aim 1). Based on our preliminary observations, we will examine the contributions of three VZV proteins, immediate early 4 (IE4), immediate early 62 (IE62) and ORF3 protein to VZV T cell tropism (Aim 2). Screening experiments that identify major VZV-induced consequences for the T cell will set the stage for additional studies of the mechanisms by which the virus achieves these effects. VZV is likely to target some pathways common to other T cell tropic viruses and strategies for applying new technologies to investigate viral pathogenesis should emerge. From a public health perspective, this work should provide information to improve the live attenuated Oka strain vaccines for varicella and zoster. While these vaccines are safe and beneficial, this work will be relevant for developing a ¿2nd generation¿ VZV vaccine which consists of a VZV recombinant virus that has impaired T cell tropism and therefore less potential to disseminate in high risk patients and to establish latency in healthy individuals.

? DESCRIPTION (provided by applicant): The ?-herpesvirus herpes simplex virus 1 (HSV-1) is a medically important pathogen in healthy individuals and immunocompromised patients. Primary infection begins with inoculation of mucosal or skin epithelial cells; the virus is then transferred by peripheral nerve axons to neurons of sensory ganglia where latency is established. HSV-1 undergoes phases of productive lytic infection and genome silencing reflecting highly regulated interactions between the virus and differentiated host cells. However, knowledge of the molecular requirements for infection and viral restriction by host cells within their distinctive tissue microenvironments is limited. We will study HSV-1 pathogenesis in human tissue, using skin xenografts in the severe combined immunodeficiency (SCID) mouse model, as a novel approach to address this gap. Our primary objective is to assess functions of the latency-associated transcript (LAT) locus. The LAT locus has been investigated extensively in HSV-1 neuropathogenesis but little is known about the contributions of LAT locus elements to productive infection in peripheral tissues. Our preliminary data show defective replication in skin of both KOS and 17syn+ mutants that carry a 203bp ?Pst core LAT promoter deletion, compared to their rescue viruses. This unexpected observation suggests that requirements for HSV-1 pathogenesis are more stringent within the human skin microenvironment. Notably, LAT functions were necessary in differentiated human skin cells in vivo even though SCID mice lack adaptive immunity. These findings warrant investigating how LAT locus transcripts and microRNAs (miRNAs) encoded in this region influence infection of human skin cells. Our approach emphasizes the opportunity to use deep sequencing methods for detecting and quantifying viral and cell mRNA transcripts and miRNAs in infected tissues, and to follow the kinetics of their expression in relation to pathogenesis in human skin in vivo. Our specific aims are designed to characterize how LAT locus transcripts (Aim 1) and LAT and cellular miRNAs produced during lytic HSV-1 infection (Aim 2) contribute to skin pathogenesis in vivo. The progression of infection is monitored using quantitative assays for viral replication and lesion formation in skin xenografts over a 3-14 day interval. These aims combine strategies of open-ended transcriptome and miRNA profiling of infected skin to analyze the effects of LAT locus mutations with the evaluation of mutants that have targeted disruptions of specific LAT miRNAs. We predict that LATs and LAT miRNAs will both regulate infection and contribute to achieving the optimal balance with intrinsic antiviral defenses, so that lesions are formed and HSV-1 can be transmitted to new hosts. More broadly, this study in the SCID xenograft model has the potential to yield key insights about HSV-1 disease not identified previously because functions encoded in the LAT region, especially long non-coding RNAs and miRNAs, are likely to be specific for the human host. From a public health perspective, understanding HSV-1 skin infection and the role of LAT locus functions is relevant for designing novel antiviral drugs or attenuated vaccines.

? DESCRIPTION (provided by applicant): Varicella-zoster virus, an ?-herpesvirus, causes varicella and zoster. Infection of T cells allows VZV transfer from the respiratory tract to skin ad the typical zoster rash results from axonal transport after VZV reactivation in neurons. Our strategy uses human tonsil T cells and skin xenografts in the SCID mouse model to investigate molecular mechanisms that mediate VZV tropism for these differentiated human cells, which are critical during pathogenesis, and the counterbalancing innate immune responses. Since VZV must infect primary host cells exhibiting their inherently stochastic states, and alter conditions o support infection within each cell, our strategy emphasizes multi-parametric single cell analyses. Our aims related to T cell tropism (Aim 1) address the hypothesis that VZV modulation of tonsil T cell mircoRNAs (miRs) regulates cell signaling networks, enhancing infection and skin homing independently of the usual TCR- mediated processes that promote T cell trafficking for antiviral immune functions, as well as other host cell factors that support replication in T cells. Single cel mass cytometry (CyTOF) will be used to relate the expression of selected miRs to changes in T cell surface and intracellular proteins and virus production in VZV-infected T cells. Contributions of VZV and host cell transactivators to miR expression will be determined. Our aims about skin tropism (Aim 2) focus on hair follicle cells as an initial site of VZV infection. Our hypothesis is that infection of follicle stem cells triggers activation and accelerates VZV delivery to interfollicular epidermal cells where lesions form, and conversely, that multifactorial IFN-related and cytokine responses of uninfected epidermal cells control the process. It will be addressed primarily by quantifying combinatorial expression of 30 intracellular and cell surface proteins in infected and uninfected skin with CyTOF. Mass spectroscopy combined with tissue section imaging for high dimensional immunohistochemistry (MIBI-TOF) using these markers will also be developed to map lesion formation in skin xenografts. These methods will be applied to determine whether age-related changes in human skin impair local innate control, contributing to severe cutaneous zoster in the elderly. Studying VZV pathogenesis is relevant for public health because new antiviral drug targets are needed, given the limited benefit of available agents. In addition, live attenuated VZV vaccines are unsafe for immunocompromised patients and can cause zoster; whether new subunit vaccines will prevent varicella is uncertain. Single cell methods also have utility for screening drug candidates for antiviral activity while revealing their potential cell toxicities. For basic research, developing approaches to assess the complex, multi-factorial effects of viruses on host cell protein networks and other regulatory elements, lik miRs, has broad applications for studying viral pathogenesis. Finally, multi-parametric single cell analyses of uninfected primary human cells should reveal aspects of T cell and skin cell biology pointing to new research directions for many purposes.

Herpesviruses are pathogens of medical and economic significance that cause a range of diseases in humans and animals. Varicella-zoster virus (VZV) is an important human alpha herpesvirus that causes varicella and zoster after reactivation from latency in sensory ganglia. The morphogenesis of varicella-zoster virus (VZV), like all herpesviruses, involves egress of DNA-containing capsids from the nucleus to the trans- Golgi network for secondary envelopment by viral glycoprotein-enriched membranes followed by transport in intracellular vesicles to the cell surface. Although purified herpesvirus structures have been described, much less is known at the structural level about virus particle morphology within infected cells and this dynamic process, which takes place at different spatial locations and temporal order. Cryogenic electron tomography (cryo-ET) has the promise to uncover 3D structures of assembly intermediates, providing structural details of each molecular component of the virion during this dynamic process. Recent advances in cryo-specimen preparation, data collection strategy, electron optics, electron detector and data processing methods make this type of study tractable. First, we will characterize the structure of VZV complete or light (L; lacking capsids) particles at the cell surface (Aim 1), based on preliminary data showing the feasibility of visualizing these particles by cryo-ET. The cryo-ET dataset will be used to derive capsid structures as a benchmark in the initial protocol development to define the attainable resolution of our data collection and image processing strategy. Next, we aim to determine the structures of the spike densities visible in our dataset, at the resolution attained for capsids. These analyses will put the glycoprotein structures in context to define their distribution, interaction with each other and possibly, structural rearrangement upon interacting with the cell surface. Second, we will characterize the structure of VZV particles inside infected cells (Aim 2). Our well- characterized VZV recombinant expressing the ORF23 capsid protein tagged with RFP will be used for correlative cryo-fluorescence confocal microscopy of vitrified cells with our new cryo-FIB/SEM instrument to prepare thin lamellae of VZV infected cells at sites of an RFP signal in the nucleus or cytoplasm. Milled lamellae will be used for cryo-ET and sub tomogram averaging to generate structures of viral and associated cellular components. This approach will be used to derive the structure of VZV capsids and associated proteins at intracellular sites, to be followed by generating de novo structures of glycoproteins on VZV particles at intracellular sites. This work will address gaps in structural knowledge of herpesvirus morphogenesis within infected cells, using VZV as a model, and advance the application of cryo-ET techniques and data analytics to the study of virus-host cell interactions, which has broad relevance including for SARS-CoV-2, and the molecular biology of human cells. These structure discoveries have the potential to inspire the development of novel drug or prophylactic strategies for the human herpesviruses.