Gag-Specific Cellular Immunity Determines In Vitro Viral Inhibition and In Vivo Virologic Control following Simian Immunodeficiency Virus Challenges of Vaccinated Rhesus Monkeys

A comprehensive vaccine for human immunodeficiency virus type 1 (HIV-1) would block HIV-1 acquisition as well as durably control viral replication in breakthrough infections. Recent studies have demonstrated that Env is required for a vaccine to protect against acquisition of simian immunodeficiency virus (SIV) in vaccinated rhesus monkeys, but the antigen requirements for virologic control remain unclear. Here, we investigate whether CD8(+) T lymphocytes from vaccinated rhesus monkeys mediate viral inhibition in vitro and whether these responses predict virologic control following SIV challenge. We observed that CD8(+) lymphocytes from 23 vaccinated rhesus monkeys inhibited replication of SIV in vitro. Moreover, the magnitude of inhibition prior to challenge was inversely correlated with set point SIV plasma viral loads after challenge. In addition, CD8 cell-mediated viral inhibition in vaccinated rhesus monkeys correlated significantly with Gag-specific, but not Pol- or Env-specific, CD4(+) and CD8(+) T lymphocyte responses. These findings demonstrate that in vitro viral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates with in vivo virologic control following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired.     

The BZLF1 homolog of an Epstein-Barr-related gamma-herpesvirus is a frequent target of the CTL response in persistently infected rhesus macaques

Although CD8(+) T lymphocytes targeting lytic infection proteins dominate the immune response to acute and persistent EBV infection, their role in immune control of EBV replication is not known. Rhesus lymphocryptovirus (rhLCV) is a gamma-herpesvirus closely related to EBV, which establishes persistent infection in rhesus macaques. In this study, we investigated cellular immune responses to the rhLCV BZLF1 (rhBZLF1) homolog in a cohort of rhLCV-seropositive rhesus macaques. rhBZLF1-specific IFN-gamma ELISPOT responses ranging between 56 and 3070 spot-forming cells/10(6) PBMC were detected in 36 of 57 (63%) rhesus macaques and were largely mediated by CD8(+) T lymphocytes. The prevalence and magnitude of ELISPOT responses were greater in adult (5-15 years of age) rather than juvenile macaques (<5 years of age), suggesting that rhBZLF1-specific CTL increase over time following early primary infection. A highly immunogenic region in the carboxyl terminus of the rhBZLF1 protein containing overlapping CTL epitopes restricted by Mamu-A*01 and other as yet unidentified MHC class I alleles was identified. The presence of a robust CD8(+) T lymphocyte response targeting this lytic infection protein in both rhesus macaques and humans suggests that these CTL may be important for immune control of EBV-related gamma-herpesvirus infection. These data underscore the utility of the rhLCV-macaque model for studies of EBV pathogenesis.     

Mice develop effective but delayed protective immune responses when immunized as neonates either intranasally with nonliving VP6/LT(R192G) or orally with live rhesus rotavirus vaccine candidates

Rotavirus vaccines are delivered early in life, when the immune system is immature. To determine the effects of immaturity on responses to candidate vaccines, neonatal (7 days old) and adult mice were immunized with single doses of either Escherichia coli-expressed rotavirus VP6 protein and the adjuvant LT(R192G) or live rhesus rotavirus (RRV), and protection against fecal rotavirus shedding following challenge with the murine rotavirus strain EDIM was determined. Neonatal mice immunized intranasally with VP6/LT(R192G) were unprotected at 10 days postimmunization (dpi) and had no detectable rotavirus B-cell (antibody) or CD4(+) CD8(+) T-cell (rotavirus-inducible, Th1 [gamma interferon and interleukin-2 {IL-2}]-, Th2 [IL-5 and IL-4]-, or ThIL-17 [IL-17]-producing spleen cells) responses. However, by 28 and 42 dpi, these mice were significantly (P >or= 0.003) protected and contained memory rotavirus-specific T cells but produced no rotavirus antibody. In contrast, adult mice were nearly fully protected by 10 dpi and contained both rotavirus immunoglobulin G and memory T cells. Neonates immunized orally with RRV were also less protected (P=0.01) than adult mice by 10 dpi and produced correspondingly less rotavirus antibody. Both groups contained few rotavirus-specific memory T cells. Protection levels by 28 dpi for neonates or adults were equal, as were rotavirus antibody levels. This report introduces a neonatal mouse model for active protection studies with rotavirus vaccines. It indicates that, with time, neonatal mice develop full protection after intranasal immunization with VP6/LT(R192G) or oral immunization with a live heterologous rotavirus and supports reports that protection depends on CD4(+) T cells or antibody, respectively.     

Immunization with baculovirus-expressed recombinant rotavirus proteins VP1, VP4, VP6, and VP7 induces CD8+ T lymphocytes that mediate clearance of chronic rotavirus infection in SCID mice.

Clearance of chronic murine rotavirus infection in SCID mice can be demonstrated by adoptive transfer of immune CD8+ T lymphocytes from histocompatible donor mice immunized with a murine homotypic rotavirus (T. Dharakul, L. Rott, and H.B. Greenberg, J. Virol 64:4375-4382, 1990). The present study focuses on the protein specificity and heterotypic nature of cell-mediated clearance of chronic murine rotavirus infection in SCID mice. Heterotypic cell-mediated clearance was demonstrated in SCID mice infected with EDIM (murine) rotavirus after adoptive transfer of CD8+ T lymphocytes from BALB/c mice that were immunized with a variety of heterologous (nonmurine) rotaviruses including Wa (human, serotype 1), SA11 and RRV (simian, serotype 3), and NCDV and RF (bovine, serotype 6). This finding indicates the serotypic independence of T-cell-mediated rotavirus clearance. To further identify the rotavirus proteins that are capable of generating CD8+ T cells that mediate virus clearance, donor mice were immunized with SF-9 cells infected with a baculovirus recombinant expressing one of the following rotavirus proteins: VP1, VP2, NS53 (from RF), VP4, VP7, NS35 (from RRV), VP6, and NS28 (from SA11). SCID mice stopped shedding rotavirus after receiving CD8+ T cells from mice immunized with VP1, VP4, VP6, and VP7 but not with VP2, NS53, NS35, NS28, or wild-type baculovirus. These results suggest that heterotypic cell-mediated clearance of rotavirus in SCID mice is mediated by three of the major rotavirus structural proteins and by a putative polymerase protein.     

Vaccination of macaques with long-standing SIVmac251 infection lowers the viral set point after cessation of antiretroviral therapy

A cohort of rhesus macaques with long-standing SIVmac251 infection (> or =5 mo) was treated with continuous antiretroviral therapy (ART). A group of eight macaques was vaccinated with or without simultaneous administration of low dose IL-2 with the highly attenuated poxvirus vector (NYVAC) vaccine candidate expressing the SIVmac structural gag-pol-env (gpe) genes and a novel chimeric fusion protein derived from the rev-tat-nef (rtn) regulatory genes. Control groups consisted of mock-vaccinated macaques or animals treated only with IL-2. Vaccination significantly expanded both virus-specific CD4(+) and CD8(+) T cell responses, and IL-2 further increased the vaccine-induced response to an immunodominant Gag epitope. Following antiretroviral treatment interruption, the viral set point was significantly lower in vaccinated than in control macaques for at least 4 consecutive mo, and viral containment was inversely correlated with vaccine-induced, virus-specific CD4(+) and CD8(+) T cell responses. These data provide the proof of concept that therapeutic vaccination before cessation of ART may be a feasible approach in the clinical management of HIV-1 infection.     

NK and CD4+ T Cell Cooperative Immune Responses Correlate with Control of Disease in a Macaque Simian Immunodeficiency Virus Infection Model

Control of infectious disease may be accomplished by successful vaccination or by complex immunologic and genetic factors favoring Ag-specific multicellular immune responses. Using a rhesus macaque model, we evaluated Ag-specific T cell-dependent NK cell immune responses in SIV-infected macaques, designated "controlling" or "noncontrolling" based on long-term chronic viremia levels, to determine whether NK cell effector functions contribute to control of SIV infection. We observed that Gag stimulation of macaque PBMCs induced subset-specific NK cell responses in SIV-controlling but not SIV-noncontrolling animals, as well as that circulatory NK cell responses were dependent on Ag-specific IL-2 production by CD4(+) central memory T cells. NK cell activation was blocked by anti-IL-2-neutralizing Ab and by CD4(+) T cell depletion, which abrogated the Gag-specific responses. Among tissue-resident cells, splenic and circulatory NK cells displayed similar activation profiles, whereas liver and mucosal NK cells displayed a decreased activation profile, similar in SIV-controlling and -noncontrolling macaques. Lack of T cell-dependent NK cell function was rescued in SIV-noncontrolling macaques through drug-mediated control of viremia. Our results indicate that control of disease progression in SIV-controlling macaques is associated with cooperation between Ag-specific CD4(+) T cells and NK cell effector function, which highlight the importance of such cell-to-cell cooperativity in adaptive immunity and suggest that this interaction should be further investigated in HIV vaccine development and other prophylactic vaccine approaches.     

Protective immunity to rotavirus shedding in the absence of interleukin-6: Th1 cells and immunoglobulin A develop normally

We investigated whether interleukin-6 (IL-6) was required for the development of immunoglobulin A (IgA)- and T-helper 1 (Th1)-associated protective immune responses to rotavirus by using adult IL-6-deficient mice [BALB/c and (C57BL/6 x O1a)F(2) backgrounds]. Naive IL-6(-) mice had normal frequencies of IgA plasma cells in the gastrointestinal tract. Consistent with this, total levels of IgA in fecal extracts, saliva, and sera were unaltered. In specific response to oral infection with rhesus rotavirus, IL-6(-) and IL-6(+) mice exhibited efficient Th1-type gamma interferon responses in Peyer's patches with high levels of serum IgG2a and intestinal IgA. Although there was an increase in Th2-type IL-4 in CD4(+) T cells from IL-6(-) mice following restimulation with rotavirus antigen in the presence of irradiated antigen-presenting cells, unfractionated Peyer's patch cells failed to produce a significant increase in IL-4. Moreover, virus-specific IgG1 in serum was not significantly increased in IL-6(-) mice in comparison with IL-6(+) mice. Following oral inoculation with murine rotavirus, IL-6(-) and IL-6(+) mice mediated clearance of rotavirus and mounted a strong IgA response. When IL-6(-) and IL-6(+) mice [(C57BL/6 x O1a)F(2) background] were orally inoculated with rhesus rotavirus and later challenged with murine rotavirus, all of the mice maintained high levels of IgA in feces and were protected against reinfection. Thus, IL-6 failed to provide unique functions in the development of IgA-secreting B cells and in the establishment of Th1-associated protective immunity against rotavirus infection in adult mice.     

Direct relationship between suppression of virus-specific immunity and emergence of cytomegalovirus disease in simian AIDS

Although opportunistic infections like cytomegalovirus (CMV) are common sequelae of end-stage AIDS, the immune events leading to CMV reactivation in human immunodeficiency virus (HIV)-infected individuals are not well defined. The role of cellular and humoral CMV-specific immune responses in immune control of latent CMV infection was evaluated prospectively in a cohort of 11 simian immunodeficiency virus (SIV)-infected CMV-seropositive rhesus macaques, 6 of whom had histologic evidence of CMV disease at death. Macaques with CMV disease differed from macaques without CMV disease in having significantly higher levels of plasma SIV RNA and CMV DNA and significantly lower titers of anti-CMV binding antibodies (Abs) at the time of death. A significant decline in anti-CMV Abs and CMV-specific CD4(+) and CD8(+) T lymphocytes over time was observed in the macaques with CMV disease, but not in the macaques without CMV disease. Reduction in CMV-specific CD8(+) T lymphocytes and anti-CMV neutralizing Abs was significantly correlated with a decline in CMV-specific CD4(+) T lymphocytes. Although declines in CMV-specific T lymphocytes alone were sufficient for reactivation of low-level CMV viremia, high-level viremia (>1,000 copies of CMV DNA per ml of plasma) was observed when anti-CMV neutralizing and binding Abs had also declined. Thus, the occurrence of CMV reactivation-associated disease in AIDS is associated with suppression of both cellular and humoral CMV-specific immune responses. The underlying mechanism may be a dysfunction of memory B and CD8(+) T lymphocytes associated with SIV-induced impairment of CMV-specific CD4(+) T-cell help.     

Development of a rotavirus-shedding model in rhesus macaques, using a homologous wild-type rotavirus of a new P genotype

Although there are several reports on rotavirus inoculation of nonhuman primates, no reliable model exists. Therefore, this study was designed to develop a rhesus macaque model for rotavirus studies. The goals were to obtain a wild-type macaque rotavirus and evaluate it as a challenge virus for model studies. Once rotavirus was shown to be endemic within the macaque colony at the Tulane National Primate Research Center, stool specimens were collected from juvenile animals (2.6 to 5.9 months of age) without evidence of previous rotavirus infection and examined for rotavirus antigen. Six of 10 animals shed rotavirus during the 10-week collection period, and the electropherotypes of all isolates were identical to each other but distinct from those of prototype simian rotaviruses. These viruses were characterized as serotype G3 and subgroup 1, properties typical of many animal rotaviruses, including simian strains. Nucleotide sequence analysis of the VP4 gene was performed with a culture-grown isolate from the stool of one animal, designated the TUCH strain. Based on both genotypic and phylogenetic comparisons between TUCH VP4 and cognate proteins of representatives of the reported 22 P genotypes, the TUCH virus belongs to a new genotype, P[23]. A pool of wild-type TUCH was prepared and intragastrically administered to eight cesarean section-derived, specific-pathogen-free macaques 14 to 42 days of age. All animals were kept in a biocontainment level 2 facility. Although no diarrhea was observed and the animals remained clinically normal, all animals shed large quantities of rotavirus antigen in their feces after inoculation, which resolved by the end of the 14-day observation period. Therefore, TUCH infection of macaques provides a useful nonhuman primate model for studies on rotavirus protection.     

Patterns of CD8+ immunodominance may influence the ability of Mamu-B*08-positive macaques to naturally control simian immunodeficiency virus SIVmac239 replication

Certain major histocompatibility complex (MHC) class I alleles are strongly associated with control of human immunodeficiency virus and simian immunodeficiency virus (SIV). CD8(+) T cells specific for epitopes restricted by these molecules may be particularly effective. Understanding how CD8(+) T cells contribute to control of viral replication should yield important insights for vaccine design. We have recently identified an Indian rhesus macaque MHC class I allele, Mamu-B*08, associated with elite control and low plasma viremia after infection with the pathogenic isolate SIVmac239. Here, we infected four Mamu-B*08-positive macaques with SIVmac239 to investigate why some of these macaques control viral replication. Three of the four macaques controlled SIVmac239 replication with plasma virus concentrations below 20,000 viral RNA copies/ml at 20 weeks postinfection; two of four macaques were elite controllers (ECs). Interestingly, two of the four macaques preserved their CD4(+) memory T lymphocytes during peak viremia, and all four recovered their CD4(+) memory T lymphocytes in the chronic phase of infection. Mamu-B*08-restricted CD8(+) T-cell responses dominated the acute phase and accounted for 23.3% to 59.6% of the total SIV-specific immune responses. Additionally, the ECs mounted strong and broad CD8(+) T-cell responses against several epitopes in Vif and Nef. Mamu-B*08-specific CD8(+) T cells accounted for the majority of mutations in the virus at 18 weeks postinfection. Interestingly, patterns of viral variation in Nef differed between the ECs and the other two macaques. Natural containment of AIDS virus replication in Mamu-B*08-positive macaques may, therefore, be related to a combination of immunodominance and viral escape from CD8(+) T-cell responses.     

Induction of mucosal homing virus-specific CD8(+) T lymphocytes by attenuated simian immunodeficiency virus

Induction of virus-specific T-cell responses in mucosal as well as systemic compartments of the immune system is likely to be a critical feature of an effective AIDS vaccine. We investigated whether virus-specific CD8(+) lymphocytes induced in rhesus macaques by immunization with attenuated simian immunodeficiency virus (SIV), an approach that is highly effective in eliciting protection against mucosal challenge, express the mucosa-homing receptor alpha4beta7 and traffic to the intestinal mucosa. SIV-specific CD8(+) T cells expressing alpha4beta7 were detected in peripheral blood and intestine of macaques infected with attenuated SIV. In contrast, virus-specific T cells in blood of animals immunized cutaneously by a combined DNA-modified vaccinia virus Ankara regimen did not express alpha4beta7. These results demonstrate the selective induction of SIV-specific CD8(+) T lymphocytes expressing alpha4beta7 by a vaccine approach that replicates in mucosal tissue and suggest that induction of virus-specific lymphocytes that are able to home to mucosal sites may be an important characteristic of a successful AIDS vaccine.     

Antigen-dependent cytokine mRNA expression by individual rhesus macaque T helper cells by flow cytometry

Specific patterns of cytokine secretion by CD4(+) T helper (Th) cells determine the nature of immune effector responses. Using a multiparameter, flow cytometric fluorescent in situ hybridization (FISH) assay that detected cytoplasmic mRNA within intact cells, we assessed antigen-specific cytokine expression in rhesus macaque Th cells. In the peripheral lymphocytes of immunized rhesus macaques, FISH detected antigen-induced cytokine gene expression in single Th cells. Analysis of simultaneous cytokine expression by single cells demonstrated that the recall immune response consisted of Th cells expressing either a Th1 (IL-2(+)/IFN-gamma(+)) or a Th2 (IL-4(+)/IL-6(+)) cytokine pattern. In addition to the classic Th subsets, Th cells expressing only one of two Th1 or Th2 defining cytokines were common following antigen restimulation. The data gathered with the FISH assay suggest that, in primates, the immune response to recall antigens consists of nonclassic Th cells, as well as a mixture of polarized Th1 and Th2 T cells.     

Potentiation of simian immunodeficiency virus (SIV)-specific CD4(+) and CD8(+) T cell responses by a DNA-SIV and NYVAC-SIV prime/boost regimen.

T cell-mediated immune responses play an important role in the containment of HIV-1 replication. Therefore, an effective vaccine against HIV-1 should be able to elicit high frequencies of virus-specific CD8(+) and CD4(+) T cells. The highly attenuated poxvirus-based vaccine candidate, NYVAC-SIV-gag-pol-env (NYVAC-SIV-gpe), has been shown to induce and/or expand SIV-specific CD4(+) and CD8(+) T cell responses in both naive and infected macaques. In this study, the immunogenicity of NYVAC-SIV-gpe alone was compared with a combination regimen where priming with an optimized DNA-SIV-gag-env vaccine candidate was followed by a NYVAC-SIV-gpe boost. In macaques immunized with the prime-boost regimen, the extent and durability of CD8(+) T cell response to an immunodominant SIV gag epitope was increased and these animals recognized a broader array of subdominant SIV epitopes in the cytolytic assay. In addition, the prime-boost regimen significantly enhanced the proliferative responses to both SIV gag and env proteins. Thus, the combination of these vaccine modalities may represent a valuable strategy in the development of a vaccine for HIV.     

Differences between T cell epitopes recognized after immunization and after infection

Evidence suggests that cellular immune responses play a crucial role in the control of HIV and SIV replication in infected individuals. Several vaccine strategies have therefore targeted these CD8(+) and CD4(+) responses. Whether vaccination induces the same repertoire of responses seen after infection is, however, a key unanswered question in HIV vaccine development. We therefore compared the epitope specificity induced by vaccination to that present postchallenge in the peripheral blood. Intracellular cytokine staining of PBMC stimulated with overlapping 15/20-mer peptides spanning the proteins of SIV were measured after DNA/modified vaccinia Ankara vaccination of eight rhesus macaques. Lymphocytes from 8 animals recognized a total of 39 CD8 epitopes and 41 CD4 epitopes encoded by the vaccine. T cell responses were again monitored after challenge with SIVmac239 to investigate the evolution of these responses. Only 57% of all CD8(+) T cell responses and 19% of all CD4(+) T cell responses present after vaccination were recalled after infection as measured in the peripheral blood. Interestingly, 29 new CD8 epitopes and 5 new CD4 epitopes were recognized by PBMC in the acute phase. These new epitopes were not detected after vaccination, and only some of them were maintained in the chronic phase (33% of CD8 and no CD4 responses). Additionally, 24 new CD8 epitopes and 7 new CD4 epitopes were recognized by PBMC in the chronic phase of infection. The repertoire of the immune response detected in the peripheral blood after immunization substantially differed from the immune response detected in the peripheral blood after infection.     

A comparison of cell-mediated immune responses in rhesus macaques housed singly, in pairs, or in groups

A variety of psychosocial factors have been shown to influence immunological responses in laboratory primates. The present investigation examined the effects of social housing condition on cell-mediated immune responses, comparing rhesus macaques (Macaca mulatta) in three housing conditions (single, pair, and group). Subjects included 12 adults of both sexes in each housing condition (N=36). Multiple blood samples (0, 4, 8, and 12 months) were collected for immunological analyses, including lymphocyte subsets, lymphocyte proliferation to pathogens and nonspecific mitogens, natural killer cell activity, and cytokine production. CD4(+) to CD8(+) ratios differed significantly across housing conditions and singly caged subjects had significantly lower CD4(+)/CD8(+) after the 4-month timepoint than did socially housed (pair and group) subjects. CD4(+) to CD8(+) ratios were positively correlated within subjects, suggesting a trait-like aspect to this parameter. Lymphocyte proliferation responses to all four gastrointestinal pathogens differed across housing conditions (at least at the 0.08 level), as did proliferation responses to StaphA, and the production of cytokines (IFN-gamma, IL-2, and IL-10). Proliferation responses of singly caged monkeys did not differ from socially housed monkeys and the highest levels of both IFN-gamma and IL-10 were produced by group housed subjects. The data demonstrate that social housing condition affects immune responses. While not unidirectional, these effects generally suggest enhanced immune responses for socially housed animals. Since rhesus monkeys live socially in nature, and the immune responses of singly housed animals differed from those housed socially, there is considerable motivation and justification for suggesting that the use of singly housed rhesus macaques may complicate interpretations of normal immunological responses. This may have important implications for the management, treatment, and selection of primate subjects for immunological studies.     

Characterization of hepatitis C virus core-specific immune responses primed in rhesus macaques by a nonclassical ISCOM vaccine

Current therapies for the treatment of hepatitis C virus (HCV) infection are only effective in a restricted number of patients. Cellular immune responses, particularly those mediated by CD8(+) CTLs, are thought to play a role in the control of infection and the response to antiviral therapies. Because the Core protein is the most conserved HCV protein among genotypes, we evaluated the ability of a Core prototype vaccine to prime cellular immune responses in rhesus macaques. Since there are serious concerns about using a genetic vaccine encoding for Core, this vaccine was a nonclassical ISCOM formulation in which the Core protein was adsorbed onto (not entrapped within) the ISCOMATRIX, resulting in approximately 1-microm particulates (as opposed to 40 nm for classical ISCOM formulations). We report that this Core-ISCOM prototype vaccine primed strong CD4(+) and CD8(+) T cell responses. Using intracellular staining for cytokines, we show that in immunized animals 0.30-0.71 and 0.32-2.21% of the circulating CD8(+) and CD4(+) T cells, respectively, were specific for naturally processed HCV Core peptides. Furthermore, this vaccine elicited a Th0-type response and induced a high titer of Abs against Core and long-lived cellular immune responses. Finally, we provide evidence that Core-ISCOM could serve as an adjuvant for the HCV envelope protein E1E2. Thus, these data provide evidence that Core-ISCOM is effective at inducing cellular and humoral immune responses in nonhuman primates.     

The rhesus rotavirus gene encoding VP4 is a major determinant in the pathogenesis of biliary atresia in newborn mice

Biliary atresia (BA) is a devastating disease of childhood for which increasing evidence supports a viral component in pathogenesis. The murine model of BA is induced by perinatal infection with rhesus rotavirus (RRV) but not with other strains of rotavirus, such as TUCH. To determine which RRV gene segment(s) is responsible for pathogenesis, we used the RRV and TUCH strains to generate a complete set of single-gene reassortants. Eleven single-gene "loss-of-function" reassortants in which a TUCH gene replaced its RRV equivalent and 11 single-gene "gain-of-function" reassortants in which an RRV gene replaced its TUCH equivalent were generated. Newborn BALB/c mice were inoculated with the reassortants and were monitored for biliary obstruction and mortality. In vitro, the ability to bind to and replicate within cholangiocytes was analyzed. Infection of mice with the "loss-of-function" reassortant R(T(VP4)), where gene 4 from TUCH was placed on an RRV background, eliminated the ability of RRV to cause murine BA. In a reciprocal fashion, the "gain-of-function" reassortant T(R(VP4)) resulted in murine BA with 88% mortality. Compared with those for RRV, R(T(VP4)) binding and titers in cholangiocytes were significantly attenuated, while T(R(VP4)) binding and titers were significantly increased over those for TUCH. Reassortants R(T(VP3)) and T(R(VP3)) induced an intermediate phenotype. RRV gene segment 4 plays a significant role in governing tropism for the cholangiocyte and the ability to induce murine BA. Gene segment 3 did not affect RRV infectivity in vitro but altered its in vivo effect.     

Administration of Recombinant Rhesus Interleukin-12 during Acute Simian Immunodeficiency Virus (SIV) Infection Leads to Decreased Viral Loads Associated with Prolonged Survival in SIVmac251-Infected Rhesus Macaques

The ability of recombinant rhesus interleukin-12 (rMamu-IL-12) administration during acute simian immunodeficiency virus SIVmac251 infection to influence the quality of the antiviral immune responses was assessed in rhesus macaques. Group I (n = 4) was the virus-only control group. Group II and III received a conditioning regimen of rMamu-IL-12 (10 and 20 microg/kg, respectively, subcutaneously [s.c.]) on days -2 and 0. Thereafter, group II received 2 microg of IL-12 per kg and group III received 10 microg/kg s.c. twice a week for 8 weeks. On day 0 all animals were infected with SIVmac251 intravenously. While all four group I animals and three of four group II animals died by 8 and 10 months post infection (p.i.), all four group III animals remained alive for >20 months p.i. The higher IL-12 dose led to lower plasma viral loads and markedly lower peripheral blood mononuclear cell and lymph node proviral DNA loads. During the acute viremia phase, the high-IL-12-dose monkeys showed an increase in CD3(-) CD8 alpha/alpha(+) and CD3(+) CD8 alpha/alpha(+) cells and, unlike the control and low-IL-12-dose animals, did not demonstrate an increase in CD4(+) CD45RA(+) CD62L(+) naive cells. The high-IL-12-dose animals also demonstrated that both CD8 alpha/alpha(+) and CD8 alpha/beta(+) cells produced antiviral factors early p.i., whereas only CD8 alpha/beta(+) cells retained this function late p.i. Long-term survival correlated with sustained high levels of SIV gag/pol and SIV env cytotoxic T lymphocytes and retention of high memory responses against nominal antigens. This is the first study to demonstrate the capacity of IL-12 to significantly protect macaques from SIV-induced disease, and it provides a useful model to more precisely identify correlates of virus-specific disease-protective responses.     

Dominance of CD8 responses specific for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection.

Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8(+) T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8(+) responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIV(mac)239 and determined all of the simian immunodeficiency virus-specific CD8(+) T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8(+) T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8(+) immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8(+) responses against Mamu-A*01-restricted epitopes Tat(28-35)SL8 and Gag(181-189)CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag(181-189)CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8(+) cellular immune response.     

B cells and TCR avidity determine distinct functions of CD4+ T cells in retroviral infection

The T cell-dependent B cell response relies on cognate interaction between B cells and CD4(+) Th cells. However, the consequences of this interaction for CD4(+) T cells are not entirely known. B cells generally promote CD4(+) T cell responses to pathogens, albeit to a variable degree. In contrast, CD4(+) T cell responses to self- or tumor Ags are often suppressed by B cells. In this study, we demonstrated that interaction with B cells dramatically inhibited the function of virus-specific CD4(+) T cells in retroviral infection. We have used Friend virus infection of mice as a model for retroviral infection, in which the behavior of virus-specific CD4(+) T cells was monitored according to their TCR avidity. We report that avidity for Ag and interaction with B cells determine distinct aspects of the primary CD4(+) T cell response to Friend virus infection. Virus-specific CD4(+) T cells followed exclusive Th1 and T follicular helper (Tfh) differentiation. High avidity for Ag facilitated expansion during priming and enhanced the capacity for IFN-γ and IL-21 production. In contrast, Tfh differentiation was not affected by avidity for Ag. By reducing or preventing B cell interaction, we found that B cells promoted Tfh differentiation, induced programmed death 1 expression, and inhibited IFN-γ production by virus-specific CD4(+) T cells. Ultimately, B cells protected hosts from CD4(+) T cell-mediated immune pathology, at the detriment of CD4(+) T cell-mediated protective immunity. Our results suggest that B cell presentation of vaccine Ags could be manipulated to direct the appropriate CD4(+) T cell response.