Pramod N. Nehete

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.

Comparison of Replication-Competent, First Generation, and Helper-Dependent Adenoviral Vaccines

All studies using human serotype 5 Adenovirus (Ad) vectors must address two major obstacles: safety and the presence of pre-existing neutralizing antibodies. Helper-Dependent (HD) Ads have been proposed as alternative vectors for gene therapy and vaccine development because they have an improved safety profile. To evaluate the potential of HD-Ad vaccines, we compared replication-competent (RC), first-generation (FG) and HD vectors for their ability to induce immune responses in mice. We show that RC-Ad5 and HD-Ad5 vectors generate stronger immune responses than FG-Ad5 vectors. HD-Ad5 vectors gave lower side effects than RC or FG-Ad, producing lower levels of tissue damage and anti-Ad T cell responses. Also, HD vectors have the benefit of being packaged by all subgroup C serotype helper viruses. We found that HD serotypes 1, 2, 5, and 6 induce anti-HIV responses equivalently. By using these HD serotypes in heterologous succession we showed that HD vectors can be used to significantly boost anti-HIV immune responses in mice and in FG-Ad5-immune macaques. Since HD vectors have been show to have an increased safety profile, do not possess any Ad genes, can be packaged by multiple serotype helper viruses, and elicit strong anti-HIV immune responses, they warrant further investigation as alternatives to FG vectors as gene-based vaccines.

Alpha-galactosylceramide is an effective mucosal adjuvant for repeated intranasal or oral delivery of HIV peptide antigens.

Mucosal delivery of vaccines against sexually transmitted pathogens is important to elicit strong immune responses at biologically relevant sites. However, inclusion of appropriate adjuvants is essential to overcome the inherent mucosal tolerance. We present evidence in support of the effectiveness of co-administering alpha-galactosylceramide (alpha-GalCer) as an adjuvant with a CTL-inducing HIV envelope peptide, via either oral or intranasal route, to prime antigen-specific immune responses in multiple systemic and mucosal compartments. Contrary to the known potential of repeated parenteral dosing with alpha-GalCer to induce NKT cell anergy that could compromise adoptive immunity development, we have observed that two and three doses delivered by the intranasal or oral route were more efficient in priming broader antigen-specific immune responses. These results demonstrate the effectiveness of alpha-GalCer as adjuvant for repeated intranasal or oral administration of vaccines for protection against mucosally transmitted pathogens.

TSLP production by epithelial cells exposed to immunodeficiency virus triggers DC-mediated mucosal infection of CD4+ T cells

Mucosal dendritic cells have been implicated in the capture, storage, and transmission of HIV to CD4+ T cells as well as in the promotion of HIV replication in activated CD4+ T cells during the cognate T-cell and DC interaction. We report that HIV induces human genital mucosal epithelial cells to produce thymic stromal lymphopoietin (TSLP) via activation of the NFκB signaling pathway. The TSLP secreted by HIV exposed epithelial cells activated DC, which promoted proliferation and HIV-1 replication of co-cultured autologous CD4+ T cells. In rhesus macaques, we observed dramatic increases in TSLP expression concurrent with an increase in viral replication in the vaginal tissues within the first 2 weeks after vaginal SIV exposure. These data suggest that HIV-mediated TSLP production by mucosal epithelial cells is a critical trigger for DC-mediated amplification of HIV-infection in activated CD4+ T cells. The cross talk between mucosal epithelial cells and DC, mediated by HIV-induced TSLP, may be an important mechanism for the high rate of HIV infection in women through the vaginal mucosa.

Rapid in vivo induction of HIV-specific cd8+ cytotoxic T lymphocytes by a 15-amino acid unmodified free peptide from the immunodominant V3-loop of GP120

Efforts to generate a vaccine to prevent infection by human immunodeficiency virus (HIV) have focused on inducing neutralizing antibodies. However, cytotoxic T-lymphocyte (CTL) responses are a major immune defense mechanism required for recovery from many different virus infections. Since CTL epitopes can be defined by short synthetic peptides, we searched for HIV peptides that elicit a viral-specific CTL response in mice. We have developed a new method for screening CTL-inducing peptides involving a single injection into the footpad of mice to prime CTLs in the draining popliteal lymph node of mice within 10 days. Our results demonstrate that a 15-amino acid peptide (aa 315-329) derived from the V3 loop of HIV gp120 caused a rapid induction of peptide-specific and gp160-specific CD8-positive CTLs. Lysis of targets is specific since cells preincubated with unrelated peptides are resistant to lysis as are cells of a different MHC haplotype pretreated with the cognate peptide. Pretreatment of restimulated node cells with complement plus anti-CD8 but not anti-CD4 removed the lytic activity. We also successfully induced in vivo CTL activity with unmodified synthetic peptides from the influenza and Sendai virus nucleoproteins, indicating general applicability of our method for rapid screening of CTL epitopes. Because HIV replication has been reported by several labs to occur mainly in lymph nodes of infected patients, the rapid induction of HIV-specific CTLs in proximal lymph nodes by unmodified peptides emphasizes the physiological significance of our findings toward vaccine and therapeutic approaches.

A post-CD4-binding step involving interaction of the V3 region of viral gp120 with host cell surface glycosphingolipids is common to entry and infection by diverse HIV-1 strains

The V3-loop region in the envelope protein gp120 of HIV is critical for viral infection, but its interaction with the target cells is not clear. Using synthetic peptides, representing linear V3 sequences as reagents, we obtained evidence to show inhibition of infection by both T-cell- and macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1) (X4 and R5, respectively), without interfering with gp120-CD4 interaction, by the V3 peptides through binding to host cell membrane glycosphingolipids (GSL). Synthetic peptides mimicking the central 15-21 amino acid sequence of the V3-loop region in both X4 and R5 strains of HIV-1 competed with and blocked the entry of both types of HIV isolates. These HIV-inhibitory V3 peptides exhibited specific binding to target cells that was not competed by antibodies to either the primary receptor CD4 or the co-receptors CXCR-4 and CCR5. However, R15K, the V3 peptide from HIV-1 IIIB gp120 exhibited specific binding to three distinct cell surface GSL: GM3, Gb3, and GalCer. Further, R15K inhibited GSL binding of gp120 from both HIV-1 IIIB (X4, Gb3-binding strain) and HIV-1 89.6 (X4R5, GM3-binding strain). Together, these results suggest a critical V3-mediated post-CD4-binding event involving cell surface GSL binding represented by the HIV-inhibitory V3 peptides, that is common for the entry of diverse HIV-1 strains and may be targeted for the development of novel HIV therapeutics aimed at blocking viral entry.

Protection against chronic infection and AIDS by an HIV envelope peptide-cocktail vaccine in a pathogenic SHIV-rhesus model

Based on our prior studies in mouse, monkey, chimpanzee, and human experimental systems, we identified six peptides encoded by highly conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope gene that selectively induce cellular immune responses in the absence of anti-viral antibody production. We tested a cocktail of the six peptides as a prototype vaccine for protection from simian human immunodeficiency virus (SHIV) infection and acquired immunodeficiency syndrome (AIDS) in a rhesus monkey model. Three monkeys were vaccinated with the peptide cocktail in Freunds adjuvant followed by autologous dendritic cells (DC) pulsed with these peptides. All the vaccinated animals exhibited significant induction of T-cell proliferation and cytotoxic T lymphocytes (CTL) responses, but no neutralizing antibodies. Two control mock-vaccinated monkeys showed no specific immune responses. Upon challenge with the pathogenic SHIV(KU-2), both the control and vaccinated monkeys were infected, but efficient clearance of virus-infected cells was observed in all the three vaccinated animals within 14 weeks. These animals also experienced a boosting of antiviral cellular immune responses after infection, and maintained antigen-specific IFN-gamma-producing cells in circulation beyond 42 weeks post-challenge. In contrast, the two mock-vaccinated monkeys had low to undetectable cellular immune responses and maintained significant levels of viral-infected cells and infectious virus in circulation. Further, in both the control monkeys plasma viremia was detectable beyond 38 weeks post-challenge indicating chronic phase infection. In one control monkey, the CD4+ cells dropped to very low levels by 2 weeks post-challenge and became undetectable by week 39 coinciding with high plasma viremia and AIDS, which included cachexia and ataxia. These results serve as proof of principle for the effectiveness of the HIV envelope peptide cocktail vaccine against chronic infection and AIDS, and support the development of multivalent peptide-based vaccine as a viable strategy to induce cell-mediated immunity (CMI) for protection against HIV and AIDS in humans.

Evidence for specific immune response against P210 BCR-ABL in long-term remission CML patients treated with interferon

Interferon-alpha treatment induces complete cytogenetic remission in 25% of Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML) patients. These remissions are durable unlike remissions induced with other therapies and yet residual leukemia is detectable in most of these patients. Total peripheral blood mononuclear cells (PBMCs) from CML patients in long-term remission following interferon treatment exhibited significantly higher proliferative responses (four- to 15-fold over background) than normals directed against P210 BCR-ABL in extracts of transfected monkey fibroblast cells. Surprisingly, similar enhanced levels of specific proliferative responses were observed with extracts from cells expressing Bcr and/or Abl proteins. In contrast, extracts from vector only or v-Mos-expressing cells had background level responses. Control monkey fibroblast cells lacking BCR-ABL expression failed to induce proliferation over background levels. Normal individuals had no significant responses to Bcr/Abl extracts. On the other hand, peripheral blood mononuclear cells from allogeneic bone marrow transplant CML patients had proliferative responses to cell extracts independent of Bcr-Abl. These data indicate that patients in remission due to alpha-interferon treatment have significantly higher levels of specific cellular immunoreactivity against Bcr/Abl sequences than normal controls, which could play a role in maintaining cytogenetic remission in Ph-positive CML patients.

Studies on in vivo induction of HIV-1 envelope-specific cytotoxic T lymphocytes by synthetic peptides from the V3 loop region of HIV-1 IIIB gp120

We have previously reported the induction of MHC class I-restricted, CD8+ cytotoxic T lymphocytes (CTLs) specific to human immunodeficiency virus type 1 (HIV-1) in mice by a 15-amino acid peptide (R15K) from the V3 loop in gp120. We now present evidence showing that CTL activity induced by R15K was stable for 8-10 weeks after a single injection and that as little as 20 micrograms peptide was sufficient for efficient CTL induction in vivo. While induction of CTLs was efficient with R15K emulsified in either complete or incomplete Freunds adjuvant, only a low-level CTL response was observed in mice immunized with R15K in either alum or saline. We analyzed a series of carrier-free synthetic peptides ranging in length from 8 to 24 amino acids from the V3 loop region and observed that peptide R10I consisting of 10 amino acids from the middle portion of R15K was more efficient for CTL induction. Additionally, lymph node cells from mice immunized with 24 and 15 amino acid peptides (N24G and R15K, respectively) when restimulated in vitro with R10I exhibited greater HIV-1 env-specific CTL activity than when either of the longer peptides was used for restimulation. A peptide consisting of only 8 amino acids (R8K) was sufficient neither for inducing primary CTLs nor for in vitro restimulation of lymph node CTL precursors. These results establish that a carrier-free 10-amino acid synthetic peptide from the V3 loop region in HIV-1 gp120 has the optimal sequence for efficient induction of HIV env-specific CTLs in mice.

Effects of dominance status and environmental enrichment on cell-mediated immunity in rhesus macaques

Abstract Psychosocial variables have been shown to affect cell-mediated immune responses in captive macaques. To explore whether one major (social dominance) and/or one minor (environmental enrichment) psychosocial variable affected immune responses in female rhesus monkeys, cell-mediated immune responses were compared in six monkeys that were the highest ranking and 12 monkeys that were middle ranking in their respective social groups. All subjects were 5- and 6-year-old rhesus living in stable unimale–multifemale groups, and were matched on social history, parity, weight, and health characteristics. Highest-ranking females had significantly lower mitogen-induced proliferation responses to lipopolysaccharide and pokeweed mitogen and higher natural killer cell activity, CD4 + lymphocyte counts, and CD8 + lymphocyte counts than did middle-ranking females. These data demonstrate that dominance rank, an important psychosocial factor, affects immune response in a stable social setting. One-half of the subjects (three highest-ranking and six middle-ranking) received a variety of environmental enhancements between the ages of 1 and 4 years (the enriched group), while the other nine subjects did not (the control group). No differences between enriched and control groups reached statistical significance, but some interesting trends appeared, tentatively suggesting that a minor psychosocial manipulation, inanimate enrichment, may subtly affect cell-mediated immune responses. The relationship between psychosocial factors and cellular immune function may have important implications for disease progression and for the management, treatment, and selection of primate subjects for studies in which immunological variables are of interest.