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Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (FcγR) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of FcγR binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to FcγR-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque FcγRIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that FcγR-mediated activities distinct from FcγRIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.

In order to elucidate the role of the inflammatory cytokines in regulating glucocorticosteroid binding (GCSB) and glucocorticosteroid receptor (GR) level we incubated a B-cell line (CESS), a promonocytic cell line (U937) and a hepatoma cell line (HepG2) in the presence of varying concentrations of IL-1β, IL-6 and TNF-α for 24 h. Glucocorticosteroid binding was determined by the method of 'whole cell uptake', and the cellular appearance of the glucocorticosteroid receptor was detected by immunocytochemistry, A rise in the glucocorticosteroid binding was induced by all three cytokines. The increase in level of glucocorticosteroid receptors in the cells shown by immunocytochemistry was much more pronounced. However, antagonistic effects were demonstrated by both methods between IL-6 and TNF-α and between IL-1β and TNF-α when they were applied simultaneously, in U937. Present data suggest that local imbalance in the ratio of these three cytokines in different pathological cases might influence the glucocorticosteroid sensitivity of the lymphocytes, monocytes and hepatocytes as target cells. [ABSTRACT FROM AUTHOR]

The vaginal epithelium of normal mice contains lymphocytes of fetal thymic origin that express an invariant Vγ4/Vδ1 TCR. The apparent lack of other γδ TCR species suggests that a selection mechanism might operate to regulate the localization of γδ T cells at this anatomical site. Selection might be connected to the Vγ4/Vδ1 TCR or to some homing characteristic of the fetal thymic lineage that appears at day 17-18 of embryonic life. In the present studies, we investigated whether transgenic γδ cells expressing a TCR species characteristic of the subpopulation of γδ T cells found in the blood, spleen and lymph would translocate to the vaginal epithelium. We found that the transgenic Vγ2 TCR+ cells did accumulate in the vagina of transgenic mice. Furthermore, like normal vaginal γδ T cells, the transgenic vaginal γδ T cells expressed the phenotype of recently activated memory/effector T cells (CD44hi, CD62L-, CD45RBlo, CD69+). Vaginal γδ T cells in normal mice do not express the CD2 and CD28 antigens, but both of these markers are present on transgenic vaginal γδ T cells. We observed that a small fraction of splenic transgenic γδ T cells had the same surface phenotype as the vaginal transgenic γδ T cells, raising the possibility that the γδ T cells present in the vaginal epithelium of transgenic mice originated from the peripheral lymphoid organs. Data in support of this possibility came from experiments in which co-incubation of splenic transgenic γδ T cells with vaginal epithelial cell suspensions induced the vaginal γδ phenotype on the splenic γδ T cells. The finding of transgenic γδ T cells in the vaginal epithelium suggests that homing of γδ T cells to this site is not restricted to γδ T cells that express the Vγ4/Vγ1 invariant TCR. Furthermore, these findings imply that retention of γδ T cells in the vaginal epithelium of normal mice is affected by a Vγ4/Vδ1-specific mechanism. The finding of a significant level of apoptosis in the transgenic vaginal γδ T cells, but not in the normal vaginal γδ T cells, could reflect that the mechanism of retention of Vγ4/Vδ1+ in the vaginal epithelium involves selective survival at the site. [ABSTRACT FROM PUBLISHER]

The effect of cytosine arabinoside on adenosylmethionine synthesis in relation to its proliferation-inhibiting ability was investigated in HT/29 and SW 620 human colon-tumor cell lines. A significant decrease in adenosylmethionine synthetase (E. C.2.4.2.13) activity was found after 2.5 h incubation with the drug, suggesting that depletion of adenosylmethionine pools might occur. Both this possible loss of adenosylmethionine and the cytostatic effect of cytosine arabinoside could partly be reversed by the exogenous administration of the former drug. Our data show that the cytostatic effect of cytosine arabinoside may be due in part to a shortage of adenosylmethionine; this finding is important for the design of combination chemotherapy regimens. [ABSTRACT FROM AUTHOR]

An abstract of the conference paper "MAb PGT121 protects against mucosal SHIV challenge in macaques at concentrations corresponding to its highly potent in vitro neutralization capacity," by B. Moldt and colleagues is presented.

The eradication of the viral reservoir represents the major obstacle to the development of a clinical cure for established HIV-1 infection. Here, we demonstrate that the administration of N-803 (brand name Anktiva) and broadly neutralizing antibodies (bNAbs) results in sustained viral control after discontinuation of antiretroviral therapy (ART) in simian-human AD8 (SHIV-AD8)-infected, ART-suppressed rhesus macaques. N-803+bNAbs treatment induced immune activation and transient viremia but only limited reductions in the SHIV reservoir. Upon ART discontinuation, viral rebound occurred in all animals, which was followed by durable control in approximately 70% of all N-803+bNAb-treated macaques. Viral control was correlated with the reprogramming of CD8 + T cells by N-803+bNAb synergy. Thus, complete eradication of the replication-competent viral reservoir is likely not a prerequisite for the induction of sustained remission after discontinuation of ART.

Protection from immunodeficiency virus challenge in nonhuman primates (NHPs) by a first-generation HIV broadly neutralizing antibody (bnAb) b12 has previously been shown to benefit from interaction between the bnAb and Fcγ receptors (FcγRs) on immune cells. To investigate the mechanism of protection for a more potent second-generation bnAb currently in clinical trials, PGT121, we carried out a series of NHP studies. These studies included treating with PGT121 at a concentration at which only half of the animals were protected to avoid potential masking of FcγR effector function benefits by dominant neutralization and using a new variant that more completely eliminated all rhesus FcγR binding than earlier variants. In contrast to b12, which required FcγR binding for optimal protection, we concluded that PGT121-mediated protection is not augmented by FcγR interaction. Thus, for HIV-passive antibody prophylaxis, these results, together with existing literature, emphasize the importance of neutralization potency for clinical antibodies, with effector function requiring evaluation for individual antibodies., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Studies have shown that vaccine vectors and route of immunization can differentially activate different arms of the immune system. However, the effects of different HIV vaccine immunogens on mucosal inflammation have not yet been studied. Because mucosal sites are the primary route of HIV infection, we evaluated the cervico-vaginal inflammatory cytokine and chemokine levels of Mauritian cynomolgus macaques following immunization and boost using two different SIV vaccine immunogens. The PCS vaccine delivers 12 20-amino acid peptides overlapping the 12 protease cleavage sites, and the Gag/Env vaccine delivers the full Gag and full Env proteins of simian immunodeficiency virus. We showed that the PCS vaccine prime and boosts induced short-lived, lower level increases of a few pro-inflammatory/chemotactic cytokines. In the PCS-vaccine group only the levels of MCP-1 were significantly increased above the baseline ( P = 0.0078, Week 6; P = 0.0078, Week 17; P = 0.0234; Week 51) following multiple boosts. In contrast, immunizations with the Gag/Env vaccine persistently increased the levels of multiple cytokines/chemokines. In the Gag/Env group, higher than baseline levels were consistently observed for IL-8 ( P = 0.0078, Week 16; P = 0.0078, Week 17; P = 0.0156, Week 52), IL-1β ( P = 0.0234, Week 16; P = 0.0156, Week 17; P = 0.0156, Week 52), and MIP-1α ( P = 0.0313, Week 16; P = 0.0156, Week 17; P = 0.0313, Week 52). Over time, repeated boosts altered the relative levels of these cytokines between the Gag/Env and PCS vaccine group. 18 weeks after final boost with a higher dosage, IP-10 levels ( P = 0.0313) in the Gag/Env group remained higher than baseline. Thus, the influence of vaccine immunogens on mucosal inflammation needs to be considered when developing and evaluating candidate HIV vaccines., (Copyright © 2020 Toledo, Li, Omange, Dacoba, Crecente-Campo, Schalk, Kashem, Rakasz, Schultz-Darken, Li, Whitney, Alonso, Plummer and Luo.)

By the end of the 2016 Zika virus (ZIKV) outbreak, it is estimated that there were up to 100 million infections in the Americas. In approximately one in seven infants born to mothers infected during pregnancy, ZIKV has been linked to microcephaly, developmental delays, or other congenital disorders collectively known as congenital Zika syndrome, as well as Guillain-Barré syndrome, in ZIKV-infected adults. It is a global health priority to develop a vaccine against ZIKV that elicits long-lasting immunity; however, the durability of immunity to ZIKV is unknown. Previous studies in mice and nonhuman primates have been crucial in vaccine development but have not defined the duration of immunity generated by ZIKV infection. In this study, we rechallenged five rhesus macaques with ZIKV 22 to 28 months after a primary ZIKV infection. We show that primary ZIKV infection generates high titers of neutralizing antibodies that protect from detectable plasma viremia following rechallenge and persist for at least 22 to 28 months. While additional longitudinal studies are necessary with longer time frames, this study establishes a new experimentally defined minimal length of protective ZIKV immunity. IMPORTANCE ZIKV emerged as a vector-borne pathogen capable of causing illness in infected adults and congenital birth defects in infants born to mothers infected during pregnancy. Despite the decrease in ZIKV cases since the 2015-2016 epidemic, questions concerning the prevalence and longevity of protective immunity have left vulnerable communities fearful that they may become the center of next ZIKV outbreak. Although preexisting herd immunity in regions of past outbreaks may dampen the potential for future outbreaks to occur, we currently do not know the longevity of protective immunity to ZIKV after a person becomes infected. Here, we establish a new experimentally defined minimal length of protective ZIKV immunity. We show that five rhesus macaques initially infected with ZIKV 22 to 28 months prior to rechallenge elicit a durable immune response that protected from detectable plasma viremia. This study establishes a new minimal length of protective immunity., (Copyright © 2020 American Society for Microbiology.)

HIV mutates rapidly and infects CD4+ T cells, especially when they are activated. A vaccine targeting conserved, essential viral elements while limiting CD4+ T cell activation could be effective. Learning from natural immunity observed in a group of highly HIV-1 exposed seronegative Kenyan female sex workers, we are testing a novel candidate HIV vaccine targeting the 12 viral protease cleavage sites (PCSs) (the PCS vaccine), in comparison with a vaccine targeting full-length Gag and Env (the Gag/Env vaccine) in a Mauritian cynomolgus macaque/SIV model. In this study we evaluated these vaccines for induction of mucosal antibodies to SIV immunogens at the female genital tract. Bio-Plex and Western blot analyses of cervicovaginal lavage samples showed that both the PCS and Gag/Env vaccines can elicit mucosal IgG antibody responses to SIV immunogens. Significantly higher increase of anti-PCS antibodies was induced by the PCS vaccine than by the Gag/Env vaccine (p<0.0001). The effect of the mucosal antibody responses in protection from repeated low dose pathogenic SIVmac251 challenges is being evaluated., Competing Interests: The authors have declared that no competing interests exist.

Developing biological interventions to control human immunodeficiency virus (HIV) replication in the absence of antiretroviral therapy (ART) could contribute to the development of a functional cure. As a potential alternative to ART, the interleukin-15 (IL-15) superagonist ALT-803 has been shown to boost the number and function of HIV-specific CD8 + T and NK cell populations in vitro Four simian immunodeficiency virus (SIV)-positive rhesus macaques, three of whom possessed major histocompatibility complex alleles associated with control of SIV and all of whom had received SIV vaccine vectors that had the potential to elicit CD8 + T cell responses, were given ALT-803 in three treatment cycles. The first and second cycles of treatment were separated by 2 weeks, while the third cycle was administered after a 29-week break. ALT-803 transiently elevated the total CD8 + effector and central memory T cell and NK cell populations in peripheral blood, while viral loads transiently decreased by ∼2 logs in all animals. Virus suppression was not sustained as T cells became less responsive to ALT-803 and waned in numbers. No effect on viral loads was observed in the second cycle of ALT-803, concurrent with downregulation of the IL-2/15 common γC and β chain receptors on both CD8 + T cells and NK cells. Furthermore, populations of immunosuppressive T cells increased during the second cycle of ALT-803 treatment. During the third treatment cycle, responsiveness to ALT-803 was restored. CD8 + T cells and NK cells increased again 3- to 5-fold, and viral loads transiently decreased again by 1 to 2 logs. IMPORTANCE Overall, our data show that ALT-803 has the potential to be used as an immunomodulatory agent to elicit effective immune control of HIV/SIV replication. We identify mechanisms to explain why virus control is transient, so that this model can be used to define a clinically appropriate treatment regimen., (Copyright © 2018 American Society for Microbiology.)

Effector memory T cell (T EM ) responses display potent antiviral properties and have been linked to stringent control of simian immunodeficiency virus (SIV) replication. Since recurrent antigen stimulation drives the differentiation of CD8 + T cells toward the T EM phenotype, in this study we incorporated a persistent herpesviral vector into a heterologous prime/boost/boost vaccine approach to maximize the induction of T EM responses. This new regimen resulted in CD8 + T EM -biased responses in four rhesus macaques, three of which controlled viral replication to <1,000 viral RNA copies/ml of plasma for more than 6 months after infection with SIVmac239. Over the course of this study, we made a series of interesting observations in one of these successful controller animals. Indeed, in vivo elimination of CD8αβ + T cells using a new CD8β-depleting antibody did not abrogate virologic control in this monkey. Only after its CD8α + lymphocytes were depleted did SIV rebound, suggesting that CD8αα + but not CD8αβ + cells were controlling viral replication. By 2 weeks postinfection (PI), the only SIV sequences that could be detected in this animal harbored a small in-frame deletion in nef affecting six amino acids. Deep sequencing of the SIVmac239 challenge stock revealed no evidence of this polymorphism. However, sequencing of the rebound virus following CD8α depletion at week 38.4 PI again revealed only the six-amino acid deletion in nef. While any role for immunological pressure on the selection of this deleted variant remains uncertain, our data provide anecdotal evidence that control of SIV replication can be maintained without an intact CD8αβ + T cell compartment.

The ability to control lentivirus replication may be determined, in part, by the extent to which individual viral proteins are targeted by the immune system. Consequently, defining the antigens that elicit the most protective immune responses may facilitate the design of effective HIV-1 vaccines. Here we vaccinated four groups of rhesus macaques with a heterologous vector prime/boost/boost/boost (PBBB) regimen expressing the following simian immunodeficiency virus (SIV) genes: env, gag, vif, rev, tat, and nef (Group 1); env, vif, rev, tat, and nef (Group 2); gag, vif, rev, tat, and nef (Group 3); or vif, rev, tat, and nef (Group 4). Following repeated intrarectal challenges with a marginal dose of the neutralization-resistant SIVmac239 clone, vaccinees in Groups 1-3 became infected at similar rates compared to control animals. Unexpectedly, vaccinees in Group 4 became infected at a slower pace than the other animals, although this difference was not statistically significant. Group 1 exhibited the best post-acquisition virologic control of SIV infection, with significant reductions in both peak and chronic phase viremia. Indeed, 5/8 Group 1 vaccinees had viral loads of less than 2,000 vRNA copies/mL of plasma in the chronic phase. Vaccine regimens that did not contain gag (Group 2), env (Group 3), or both of these inserts (Group 4) were largely ineffective at decreasing viremia. Thus, vaccine-induced immune responses against both Gag and Env appeared to maximize control of immunodeficiency virus replication. Collectively, these findings are relevant for HIV-1 vaccine design as they provide additional insights into which of the lentiviral proteins might serve as the best vaccine immunogens.

Measures to prevent sexual mucosal transmission are critically needed, particularly to prevent transmission to young women at high risk in the microepidemics in South Africa that disproportionally contribute to the continued pandemic. To that end, microbicides containing anti-retroviral (ARV) agents have been shown to prevent transmission, but with efficacy limited both by adherence and pre-existing innate immune and inflammatory conditions in the female reproductive tract (FRT). Glycerol monolaurate (GML) has been proposed as a microbicide component to enhance efficacy by blocking these transmission-facilitating innate immune response to vaginal exposure. We show here in an especially rigorous test of protection in the SIV-rhesus macaque model of HIV-1 transmission to women, that GML used daily and before vaginal challenge protects against repeat high doses of SIV by criteria that include virological and immunological assays to detect occult infection. We also provide evidence for indirect mechanisms of action in GML-mediated protection. Developing a sustained formulation for GML delivery could contribute an independent, complementary protective component to an ARV-containing microbicide.

Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV) replication has not been found to date. In this study, CD8(+) T-cell cytotoxic capacity was examined to determine whether this function is a correlate of immune control in the rhesus macaque (RM) SIV infection model as has been suggested in chronic HIV infection. SIVmac251-infected human reverse transcriptase (hTERT)-transduced CD4(+) T-cell clone targets were co-incubated with autologous macaque effector cells to measure infected CD4(+) T-cell elimination (ICE). Twenty-three SIV-infected rhesus macaques with widely varying plasma viral RNA levels were evaluated in a blinded fashion. Nineteen of 23 subjects (83%) were correctly classified as long-term nonprogressor/elite controller (LTNP/EC), slow progressor, progressor or SIV-negative rhesus macaques based on measurements of ICE (weighted Kappa 0.75). LTNP/EC had higher median ICE than progressors (67.3% [22.0-91.7%] vs. 23.7% [0.0-58.0%], p = 0.002). In addition, significant correlations between ICE and viral load (r = -0.57, p = 0.01), and between granzyme B delivery and ICE (r = 0.89, p<0.001) were observed. Furthermore, the CD8(+) T cells of LTNP/EC exhibited higher per-cell cytotoxic capacity than those of progressors (p = 0.004). These findings support that greater lytic granule loading of virus-specific CD8(+) T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. Therefore, such measurements appear to represent a correlate of control of viral replication in chronic SIV infection and their role as predictors of immunologic control in the vaccine setting should be evaluated.

Developing a vaccine for human immunodeficiency virus (HIV) may be aided by a complete understanding of those rare cases in which some HIV-infected individuals control replication of the virus. Most of these elite controllers express the histocompatibility alleles HLA-B*57 or HLA-B*27 (ref. 3). These alleles remain by far the most robust associations with low concentrations of plasma virus, yet the mechanism of control in these individuals is not entirely clear. Here we vaccinate Indian rhesus macaques that express Mamu-B*08, an animal model for HLA-B*27-mediated elite control, with three Mamu-B*08-restricted CD8(+) T-cell epitopes, and demonstrate that these vaccinated animals control replication of the highly pathogenic clonal simian immunodeficiency virus (SIV) mac239 virus. High frequencies of CD8(+) T cells against these Vif and Nef epitopes in the blood, lymph nodes and colon were associated with viral control. Moreover, the frequency of the CD8(+) T-cell response against the Nef RL10 epitope (Nef amino acids 137-146) correlated significantly with reduced acute phase viraemia. Finally, two of the eight vaccinees lost control of viral replication in the chronic phase, concomitant with escape in all three targeted epitopes, further implicating these three CD8(+) T-cell responses in the control of viral replication. Our findings indicate that narrowly targeted vaccine-induced virus-specific CD8(+) T-cell responses can control replication of the AIDS virus.

The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.

Passive transfer studies using monoclonal or polyclonal antibodies in the macaque model have been valuable for determining conditions for antibody protection against immunodeficiency virus challenge. Most studies have employed hybrid simian/human immunodeficiency virus (SHIV) challenge in conjunction with neutralizing human monoclonal antibodies. Passive protection against SIV, particularly the pathogenic prototype virus SIVmac239, has been little studied because of the paucity of neutralizing antibodies to this virus. Here, we show that the antibody-like molecule CD4-IgG2 potently neutralizes SIVmac239 in vitro. When administered by an osmotic pump to maintain concentrations given the short half-life of CD4-IgG2 in macaques, the molecule provided sterilizing immunity/protection against high-dose mucosal viral challenge to a high proportion of animals (5/7 at a 200 mg dose CD4-IgG2 and 3/6 at a 20 mg dose) at serum concentrations below 1.5 µg/ml. The neutralizing titers of such sera were predicted to be very low and indeed sera at a 1:4 dilution produced no neutralization in a pseudovirus assay. Macaque anti-human CD4 titers did develop weakly at later time points in some animals but were not associated with the level of protection against viral challenge. The results show that, although SIVmac239 is considered a highly pathogenic virus for which vaccine-induced T cell responses in particular have provided limited benefit against high dose challenge, the antibody-like CD4-IgG2 molecule at surprisingly low serum concentration affords sterilizing immunity/protection to a majority of animals.

The goal of an AIDS vaccine regimen designed to induce cellular immune responses should be to reduce the viral set point and preserve memory CD4 lymphocytes. Here we investigated whether vaccine-induced cellular immunity in the absence of any Env-specific antibodies can control viral replication following multiple low-dose challenges with the highly pathogenic SIVmac239 isolate. Eight Mamu-A*01-positive Indian rhesus macaques were vaccinated with simian immunodeficiency virus (SIV) gag, tat, rev, and nef using a DNA prime-adenovirus boost strategy. Peak viremia (P = 0.007) and the chronic phase set point (P = 0.0192) were significantly decreased in the vaccinated cohort, out to 1 year postinfection. Loss of CD4(+) memory populations was also ameliorated in vaccinated animals. Interestingly, only one of the eight vaccinees developed Env-specific neutralizing antibodies after infection. The control observed was significantly improved over that observed in animals vaccinated with SIV gag only. Vaccine-induced cellular immune responses can, therefore, exert a measure of control over replication of the AIDS virus in the complete absence of neutralizing antibody and give us hope that a vaccine designed to induce cellular immune responses might control viral replication.

In the acute stage of infection following sexual transmission of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), virus-specific CD8+ T-lymphocyte responses partially control but do not eradicate infection from the lymphatic tissues (LTs) or prevent the particularly massive depletion of CD4+ T lymphocytes in gut-associated lymphatic tissue (GALT). We explored hypothetical explanations for this failure to clear infection and prevent CD4+ T-lymphocyte loss in the SIV/rhesus macaque model of intravaginal transmission. We examined the relationship between the timing and magnitude of the CD8+ T-lymphocyte response to immunodominant SIV epitopes and viral replication, and we show first that the failure to contain infection is not because the female reproductive tract is a poor inductive site. We documented robust responses in cervicovaginal tissues and uterus, but only several days after the peak of virus production. Second, while we also documented a modest response in the draining genital and peripheral lymph nodes, the response at these sites also lagged behind peak virus production in these LT compartments. Third, we found that the response in GALT was surprisingly low or undetectable, possibly contributing to the severe and sustained depletion of CD4+ T lymphocytes in the GALT. Thus, the virus-specific CD8+ T-lymphocyte response is "too late and too little" to clear infection and prevent CD4+ T-lymphocyte loss. However, the robust response in female reproductive tissues may be an encouraging sign that vaccines that rapidly induce high-frequency CD8+ T-lymphocyte responses might be able to prevent acquisition of HIV-1 infection by the most common route of transmission.

Simian immunodeficiency virus (SIV) infection of rhesus macaques provides an excellent model for investigating the basis of protective immunity against human immunodeficiency virus (HIV). One limitation of this model, however, has been the availability of a small number of known MHC class I-restricted CTL epitopes for investigating virus-specific immune responses. We assessed CTL responses against SIV Gag in a cohort of DNA/modified vaccinia virus Ankara (MVA)-vaccinated/simian-human immunodeficiency virus (SHIV)-challenged rhesus macaques. Here, we report the identification of five novel SIV CTL epitopes in Gag for the first time (Gag(39-46) NELDRFGL, Gag(169-177) EVVPGFQAL, Gag(198-206) AAMQIIRDI, Gag(257-265) IPVGNIYRR and Gag(296-305) SYVDRFYKSL) that are restricted by the common MHC class I molecule Mamu-B*01. CTL responses to these epitopes were readily detected in cryopreserved PBMC in multiple animals up to 62 weeks post-infection, both by IFN-gamma enzyme-linked immunospot assay and intracellular IFN-gamma staining. Importantly, viral sequencing results revealed that these epitopes are highly conserved in the SIV-challenged macaques over a long period of time, indicating functional constraints in these regions. Moreover, the presence of CTL responses targeting these epitopes has been confirmed in two independent cohorts of rhesus macaques that have been challenged by SHIV or SIV. Our findings provide valuable candidates for poly-epitope vaccines and for long-term quantitative monitoring of epitope-specific CD8(+) responses in the context of this common Mamu class I allele. It may thus help increase the supply of rhesus macaques in which epitope-specific immunity can be studied in the context of SIV vaccine design.

Sexually transmitted diseases (STD) are now considered to be among the most common human infections. The incidence of STD is on the rise, which is partly due to frequent transmission during the asymptomatic phase of infection. The compounded cost of STD just in the United States is estimated to exceed $10 billion annually. STD are particularly prevalent in teenagers and young adults and the health problems caused by these diseases tend to be more severe and more frequent in woman than in men. Despite considerable efforts, a vaccine that provides protective immunity against sexually transmitted diseases in humans has not been developed. Nonetheless, research in animal models indicates that strong local and regional immune responses can influence the outcome of vaginal challenge with microbial pathogens. Vaginal immunity is an area of basic immunology that has received relatively little attention, but it is already clear that the mucosal and regional immunology of the vagina has unique features. The present review summarizes some of the anatomical, physiological and immunological features of the vagina and uterus that distinguish humans, non-human primates, rats and mice. These interspecies differences need to be taken into account in laboratory efforts to develop effective vaccines for STD in humans.

Gammadelta T cells show preferential homing that is characterized by biased TCR repertoire at different anatomical locations. The processes that regulate this compartmentalization are largely unknown. A model that allows repeated multiple sample procurement under different conditions and enables with relatively straightforward extrapolation to a human situation will facilitate our understanding. The peripheral blood Vgamma2 T cell population is the best-characterized human gammadelta T cell subset. To determine its diversity at multiple immunocompartments matching blood, colon, and vagina samples from rhesus macaques were investigated. Four joining segments used in Vgamma2-Jgamma transcripts were identified, including one segment with no human counterpart. Like in humans, the rhesus peripheral blood Vgamma2 TCR repertoire was limited and contained common sequences that were shared by genetically heterogeneous animals. Furthermore, this subset comprised several phylogenetically conserved Vgamma2 complementarity-determining region 3 (CDR3) motifs between rhesus and humans. Common sequences were also found within the colon and vagina of the same animal, and within the peripheral blood and intestine of different unrelated animals. These results validate rhesus macaques as a useful model for gammadelta TCR repertoire and homing studies. Moreover, they provide evidence that the concept of limited but overlapping Vgamma TCR repertoire between unrelated individuals can be extended including the mucosa of the digestive and reproductive tract.

Although their precise roles are not well defined, gammadelta T lymphocytes are recognized as regular components of immune responses. These cells express a limited T cell receptor repertoire and they can be stimulated by soluble ligands without conventional processing and presentation by major histocompatibility antigens. Progress in this area has been limited by the substantial differences between murine and human gammadelta T cells and the lack of knowledge about these cells in nonhuman primates. We used molecular analysis of T cell receptor diversity to characterize gammadelta T cell populations from peripheral blood and colon of rhesus macaques (Macaca mulatta). The gammadelta T cell receptor diversity was limited and distinct for these tissue compartments, particularly in the TCRGV2 family. Furthermore, the TCRDV1 + subset of peripheral blood gammadelta T cells showed signs of progressive oligoclonalization as a function of age. Similar observations have been reported for human tissue samples and our results validate rhesus macaques as an appropriate animal model for studying primate gammadelta T cell populations.

Background: Until recently, there were no CD3 antibodies that crossreacted with rhesus macaque T cells. Consequently, studies relying on CD8 counts or CD4/CD8 ratios enumerated this subpopulation on the basis of CD8+ or CD8bright+ staining. We used a rhesus-specific, anti-CD3 antibody to better define the CD8+ T-cell population, and to show the effects of better measurements on CD4/CD8 ratios and changes in T cells as macaques age., Methods: We used three-color flow cytometry to measure CD4 and CD8 populations with and without CD3 costaining. Venous blood samples were obtained from 52 colony-bred macaques between 2 months and 9 years of age., Results: The CD8+ T cells defined by CD3 and CD8 double staining were approximately 60% of all cells that were stained by CD8 alone. Improved detection of this lymphocyte subset showed that CD4/CD8 ratios were close to the range of 1.5-2.0. Declining CD4/CD8 ratios during aging are predominantly due to decreasing CD4+ T-cell counts., Conclusions: Better quantitation of the CD8+ T-cell population showed that the CD4/CD8 ratio was not inverted as had been reported, but is actually very similar to the values observed in human beings. Although the two species differ in the pattern of CD8 expression, the general immune system characteristics are very similar., (Copyright 2000 Wiley-Liss, Inc.)

The purpose of the present studies was to determine whether acute vaginal infection with Herpes virus 2 altered the vaginal population of gammadelta T cells, and whether gammadelta T cells influenced the vaginal clearance of HSV-2. BALB/c mice were infected intravaginally with the progressively lethal wild type 333 strain, or the non-lethal thymidine kinase deficient (deltaTK- -HSV-2) mutant strain of HSV-2 virus. Changes in vaginal T cell composition were examined by FACS analysis 4 days after infection. Clearance of vaginal deltaTK- -HSV-2 infection was compared between mice with normal gammadelta T cell populations (BALB/c) and transgenic mice in which all the gammadelta T cells express a receptor that is specific for the b allotype of MHC class Ib T10 antigen (G8/BALB/c). In HSV-2 infected BALB/c mice, but not G8/BALB/c, a subset of gammadelta T cells that express a Vgamma2 TCR accumulated in the vaginal mucosa by the fourth day after infection. Unexpectedly, we found that gammadelta TCR transgenic mice exhibited a more rapid clearance of the virus than control mice (P < 0.05). These findings argue against the hypothesis that the normal populations of vaginal intraepithelial gammadelta T cells play a direct role in the elimination of virally infected epithelial cells.

Schistosome granulomas from normal or IL-4-deficient C57BL/6 mice make little IFN-gamma and show no Th1 polarization. This could signify that these granulomas have few cells capable of IFN-gamma synthesis or that such cells are under tight control. Granulomas can make IL-10 and TGF-beta, which can regulate IFN-gamma synthesis. Using FACS analysis and ELISA, we explored the origin and regulation of IFN-gamma in schistosome granulomas from both IL-4(-/-) and IL-4(+/+) mice. FACS analysis of intracytoplasmic IFN-gamma staining showed that some granuloma Thy1.2+ T cells (CD8+ and CD4+) express IFN-gamma. Granulomas had NK1.1+ cells, but they appeared to produce little or no IFN-gamma. Purified granuloma Thy1.2+ cells made IFN-gamma in vitro, whereas isolated NK1.1+ lymphocytes secreted little even with rIL-12 stimulation. Culture of granuloma cells with blocking anti-IL-10 or anti-TGF-beta mAb or with rIL-12 substantially increased T cell IFN-gamma synthesis, particularly in the IL-4(-/-) animals. Cultured granuloma cells depleted of Thy1.2+ lymphocytes by Ab and C released no IFN-gamma. It is concluded that granuloma IFN-gamma comes from T cells, not NK cells. Also, this T cell-derived IFN-gamma is subject to IL-10 and TGF-beta regulation, which is particularly evident in IL-4(-/-) mice. Thus, the Th2 granuloma of schistosomiasis has large numbers of activated Th1 or Th0 lymphocytes that are under tight restraint.

Chronic infection with Schistosoma mansoni induces in humans and mice a Th2-dominant immune response in which eosinophils and IgE are conspicuously elevated. Human eosinophils express IgE receptors that participate in an IgE-dependent eosinophil-mediated ADCC reaction against Schistosomula larvae in vitro. To investigate the expression of IgE receptors on murine eosinophils, they were purified (>95% pure by Giemsa-stained cytospin preparations) from liver granulomas of Schistosoma-infected mice. Flow cytometric analysis showed the absence of the low-affinity IgE receptor Fc-epsilon RII (CD23) and Mac-2 and the absence of binding of murine IgE. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of granuloma eosinophil mRNA did not detect transcripts for Fc-epsilon RII or the alpha-chain of the high-affinity IgE receptor Fc-epsilon RI, but did detect transcripts that encode Mac-2 and the low-affinity IgG receptors Fc-gamma RIIb2, Fc-gamma RIII, and the FcR-associated gamma-chain. In vitro stimulation of granuloma eosinophils with interleukin-4 (IL-4) did not induce IgE binding, surface expression of Mac-2, or the transcription of Fc-epsilon receptors (Fc-epsilon RI, Fc-epsilon RII/CD23). To investigate normal murine eosinophils, we cultured normal mouse bone marrow cells with recombinant IL-3, recombinant IL-5, and recombinant granulocyte-macrophage colony-stimulating factor, conditions that promote eosinophil differentiation. Flow cytometric analysis of bone marrow-derived eosinophils failed to detect IgE binding or cell surface expression of Fc-epsilon RII and Mac-2, and RT-PCR analysis of fluorescence-activated cell sorted bone marrow-derived eosinophils failed to detect transcripts that encode Fc-epsilon RI or Fc-epsilon RII. These findings show that, in contrast to human eosinophils, murine eosinophils do not express cell surface receptors that bind IgE. However, because IgG receptors (Fc-gamma RIIb2, Fc-gamma RII) were present on eosinophils purified from granulomas, we investigated whether they might be involved in eosinophil activation. We found that an oxidative burst in eosinophils could be triggered through their IgG receptors.

The epithelium of the murine vagina contains a resident population of gamma delta T-cells that expresses a homogenous Vgamma4/Vdelta1 TCR lacking N-region junctional diversity, implying that these T-cells recognize a very limited array of antigenic structures. The vaginal gamma delta T-cells express a pattern of surface markers characteristic of memory/effector T-cells that have previously been activated. Although vaginal gamma delta T-cells do not express the major costimulatory molecules CD28 and CD2, they do proliferate in response to a systemically delivered anti gamma delta TCR stimulus. Vaginal gamma delta T-cells contain mRNA that encodes the keratinocyte growth factor raising the possibility that these cells play a role in the repair of vaginal epithelium following injury. While the antigen recognized by the vaginal gamma delta TCR is unknown, a model is proposed which attempts to relate some of the unusual phenotypic features of vaginal gamma delta T-cells to the physiological injury and shedding of vaginal epithelium that occurs during the estrous cycle.

In order to elucidate the role of the inflammatory cytokines in regulating glucocorticosteroid binding (GCSB) and glucocorticosteroid receptor (GR) level we incubated a B-cell line (CESS), a promonocytic cell line (U937) and a hepatoma cell line (HepG2) in the presence of varying concentrations of IL-1 beta, IL-6 and TNF-alpha for 24 h. Glucocorticosteroid binding was determined by the method of 'whole cell uptake', and the cellular appearance of the glucocorticosteroid receptor was detected by immunocytochemistry. A rise in the glucocorticosteroid binding was induced by all three cytokines. The increase in level of glucocorticosteroid receptors in the cells shown by immunocytochemistry was much more pronounced. However, antagonistic effects were demonstrated by both methods between IL-6 and TNF-alpha, and between IL-1 beta and TNF-alpha when they were applied simultaneously, in U937. Present data suggest that local imbalance in the ratio of these three cytokines in different pathological cases might influence the glucocorticosteroid sensitivity of the lymphocytes, monocytes and hepatocytes as target cells.