Gag Peptides Research Articles

The recognition of HIV-1 consensus group M Gag and Nef peptide reagents in mono- and multi-clade epidemics: implications for HIV vaccine design

Background The high level of genetic diversity of HIV-1 poses a major challenge for global vaccine development. Vaccines based on centralized sequences would minimize genetic distances to multiple clades and potentially maximize crossreactivity. Whether reactivity of these centralized peptide reagents differs in mono- and multi-clade epidemic is unknown. Methods In this study, full-length gag and nef gene sequences (from Cameroon, 50 and 54, respectively and South Africa, 23 and 19, respectively) were characterized. HIV-specific T-cell responses to group M consensus Gag and Nef peptide reagents were characterized at the peptide level using the IFN-g ELISpot assay. Results Viruses from Cameron exhibited a large degree of genetic diversity; all subtypes were present excluding subtype C, with CRF02_AG being dominating (49%). Contrary, sequenced viruses from South Africa were all pure subtype C viruses. Despite the greater diversity of viral clades in the Cameroonian cohort, the genetic distances to the consensus M reagents was similar for both cohorts (11% and 15% for Gag and Nef, respectively). Whilst the magnitude and breadth of responses to Con M Gag and Nef did not differ significantly between the two cohorts, there was a greater frequency of responders in the Cameroonian cohort compared to South Africans (95% versus 82%, respectively). For the Cameroonian cohort, 75/182 (41%) of the consensus M peptides were targeted, while for the South African cohort 66/182 (36%) of the peptides were targeted, with the majority being recognized in only 1-2 participants (69% and 76%, respectively). Patterns of immunodominance and targeting, however, differed dramatically between the two cohorts, with only 36% and 39% of Gag and Nef peptides commonly recognized between the two cohorts. Conclusion Although similarities in total magnitude and breadth may be observed between different epidemics, patterns of immunodominance of centralized immunogens may be different which may have implications for vaccine development.

Control of HIV-1 by multiple immunodominant HIV-1-specific CD8+ T cells in HIV-1-infected Japanese individuals

Background Previous studies of the comprehensive analysis of HIV-1specific CTL responses in Caucasian and African cohorts demonstrated the association of the CTL responses to HIV-1 Gag protein with the control of HIV-1 replication. However, such analysis in Asian cohorts has not been reported. In the present study, we performed the comprehensive analysis of CD8 + T cell responses against 11-mer overlapping HIV-1 Nef, Gag, and Pol peptides in 401 chronically HIV-1 clade B-infected treatment-naive Japanese individuals. Methods The CD8 + T cell responses to cocktails of the peptides were evaluated by measuring IFN-g-producing CD8 + T cells by using ELISPOT assay. Results To clarify CTLs which control HIV-1 infection in this cohort, we statistically analyzed differences of viral load and CD4 counts between responders to each peptide cocktail in each HLA + individuals and non-responders using two-tailed Mann-Whitney’s test. We found that several HLA alleles were significantly correlated with low viral load and high CD4 counts in the responses to 5 Nef, 10 Gag, or 16 Pol cocktails. In these cocktails, we identified 2 Nef, 12 Gag and 7 Pol CTL epitopes restricted by 9 HLA alleles. The breadth of CTL responses to these epitopes was significantly associated with low viral load (p=1.7x10 -10 ) and high CD4 counts (p=4.1x10 -13 ). The total magnitude of responses to the epitopes was also

Mapping of SIVmac T-cell epitopes in cynomolgus macaques immunized with auxo-GTU-MultiSIV DNA by the intradermal route followed by electroporation

Methods Naive male mauritian cynomolgus macaques were immunized with auxo-GTU-MultiSIV DNA encoding Gag, Nef, Rev and Tat (n=14) or by a combination of auxo-GTUMultiSIV DNA and auxo-GTU-SIV-vifvprvpx (n=12) by the intradermal route followed by electroporation. MHC class I and class II haplotypes were determined by microsatellite analysis. T-cell epitopes were mapped by IFN-g ELISPOT in PBMC by using matrix of 15-mer (overlapping by 11) SIVmac239 Gag, Nef, Rev, Tat, Vif, Vpx and Vpr peptides. Sequence and length of candidate epitopes were further optimized.

Induction of HIV-1 Gag-specific memory T cells in Chacma baboons by MVA prime and VLP boost vaccine regimen

Methods Peripheral blood mononuclear cells (PBMC) from baboons primed with MVA and boosted with VLPs (n=3) or vaccinated with VLPs only controls (n=2) were stimulated with HIV-1 Gag peptide pools. T cell cytokine production (multiplex TNF-a, IFN-y and IL-2) and memory phenotype was determined by flow cytometry. Human anti-CD28 and CD95 antibodies were used to delineate effector memory (Tem) and central memory (Tcm) T cells.

Immune response after vaccination of HIV infected individuals receiving HAART with overlapping gag peptides pulsed on autologous cells

Methods We performed a phase I, single centre, placebo-controlled, double-blinded and dose-escalating study to evaluate the safety and preliminary immunogenicity of a novel vaccine approach Opal-HIV-Gag(c). This vaccine is constituted by 120 15mer peptides, overlapping by 11 amino acids spanning the entire HIV Gag C Durban consensus sequence proteome, pulsed on white blood cells enriched from whole blood using a closed system, followed by intravenously reinfusion. Patients with well controlled HIV on HAART received four vaccinations administered at week 0, 4, 8 and 12, and were followed up for 12 weeks posttreatment. Eighteen people were enrolled in three groups: 12mg (n=6), 24mg (n=6) or matching placebo (n=6). An additional group (48mg, n=2) was not evaluable. Immunogenicity was assessed by IFNg ELIspot/ICS.

PD-1, IL-10, IFN-γ and IL-12 form a network to regulate HIV-1-specific CD4 T Cell and antigen-presenting cell function

Background PD-1 and IL-10 blockade can restore antigen-specific T cell functions in chronic infections and cancer. However, not all subjects respond to inhibition of either pathway, the potential differences in functions restored by these interventions are unknown, and mechanistic interactions between these pathways are poorly understood. Methods We investigated 45 subjects with HIV-1 infection with different disease status. We used CFSE assays to measure proliferation of HIV-1-specific CD4 T cells and Luminex arrays to analyze IFN-g(Th1), IL-2(Th0), IL-13(Th2) and IL-12 secretion in supernatants of CD8-depleted PBMC stimulated for 48h with Gag peptide pools in the presence of isotype control antibody, anti-PD-L1 and/or anti-IL-10Ra, anti-IFN-g or anti-IL-12. We used flow cytometry to evaluate the role of IFN-g in regulating PD-L1, HLA-DR, HLA-ABC and CD86 expression by monocytes.

First-in-human phase I clinical trial of a recombinant vesicular stomatitis virus (rVSV)-based preventive HIV-1 vaccine

Methods HVTN 090 enrolled sixty healthy, HIV-1-uninfected adults in a randomized, double-blinded, placebo-controlled dose escalation study. Groups of 12 participants received rVSV Indiana HIV Gag vaccine at 5 dose levels (4.6 x 10 to 3.4 x 10 PFU) (N=10/group) or placebo (N=2/group), delivered intramuscularly at 0 and 2 months. Reactogenicity over 7 days, adverse events (AEs), and viral cultures from whole blood, urine, saliva and swabs of oral lesions were collected. HIV-1-specific CD4+ and CD8+ T-cell responses to Gag peptides were measured 1 and 2 weeks post-boost by intracellular cytokine staining.

A new method for integrated analysis applied to gene expression and cytokines secretion in response to LIPO-5 vaccine in HIV-negative volunteers

Methods The statistical approach combines multilevel and multivariate analyses. The statistical approach is based on a variance decomposition followed by a sparse Partial Least Square Discriminant Analysis (sPLS-DA) to select the discriminative features (genes, cytokines) separating the classes in a supervised framework, or sPLS to select subsets of correlated features (genes and cytokines) in an integrative framework. HIV-LIPO-5 vaccine is a mix of five synthetic HIV-1 peptides (from Gag, Pol, Nef), each coupled to a palmytoil tail. PBMCs from 12 HIV-negative volunteers were collected before (W0) and after vaccination (W14). PBMC were stimulated with either i) the HIV-LIPO-5 vaccine; or ii) a pool of 15-mer Gag peptides included in the HIV-LIPO-5 vaccine (Gag+); or iii) a pool of 15mer peptides not included in LIPO-5 vaccine (Gag-). Production of 10 cytokines was assessed at day 11 (MILLIPLEX MAP kit, Millipore). Gene transcription in PBMC was assessed after 6and 24-hour stimulations (Illumina Human HT12-v4 chips).

HIV Controllers Maintain a Population of Highly Efficient Th1 Effector Cells in Contrast to Patients Treated in the Long Term

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral therapy. To identify parameters of the CD4 response that may contribute to viral control rather than merely reflect a persistently low viremia, we compared the T helper profiles in two groups of patients with more than 10 years of viral suppression: HIV controllers from the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) CO18 cohort (n = 26) and efficiently treated patients (n = 16). Cells specific for immunodominant Gag and cytomegalovirus (CMV) peptides were evaluated for the production of 10 cytokines and cytotoxicity markers and were also directly quantified ex vivo by major histocompatibility complex (MHC) class II tetramer staining. HIV controller CD4(+) T cells were characterized by a higher frequency of gamma interferon (IFN-γ) production, perforin(+)/CD107a(+) expression, and polyfunctionality in response to Gag peptides. While interleukin 4 (IL-4), IL-17, and IL-21 production did not differ between groups, the cells of treated patients produced more IL-10 in response to Gag and CMV peptides, pointing to persistent negative immunoregulation after long-term antiretroviral therapy. Gag293 tetramer-positive cells were detected at a high frequency (0.12%) and correlated positively with IFN-γ-producing CD4(+) T cells in the controller group (R = 0.73; P = 0.003). Tetramer-positive cells were fewer in the highly active antiretroviral therapy (HAART) group (0.04%) and did not correlate with IFN-γ production, supporting the notion of a persistent immune dysfunction in HIV-specific CD4(+) T cells of treated patients. In conclusion, HIV controllers maintained a population of highly efficient Th1 effectors directed against Gag in spite of a persistently low antigenemia, while patients treated in the long term showed a loss of CD4 effector functions.

HLA-Cw*0102-restricted HIV-1 p24 epitope variants can modulate the binding of the inhibitory KIR2DL2 receptor and primary NK cell function.

Accumulating evidence suggests an important role for Natural Killer (NK) cells in the control of HIV-1 infection. Recently, it was shown that NK cell-mediated immune pressure can result in the selection of HIV-1 escape mutations. A potential mechanism for this NK cell escape is the selection of HLA class I-presented HIV-1 epitopes that allow for the engagement of inhibitory killer cell immunoglobulin-like receptors (KIRs), notably KIR2DL2. We therefore investigated the consequences of sequence variations within HLA-Cw*0102-restricted epitopes on the interaction of HLA-Cw*0102 with KIR2DL2 using a large panel of overlapping HIV-1 p24 Gag peptides. 217 decameric peptides spanning the HIV-1 p24 Gag consensus sequence were screened for HLA-Cw*0102 stabilization by co-incubation with Cw*0102(+)/TAP-deficient T2 cells using a flow cytometry-based assay. KIR2DL2 binding was assessed using a KIR2DL2-IgG fusion construct. Function of KIR2DL2(+) NK cells was flow cytometrically analyzed by measuring degranulation of primary NK cells after co-incubation with peptide-pulsed T2 cells. We identified 11 peptides stabilizing HLA-Cw*0102 on the surface of T2 cells. However, only one peptide (p24 Gag209–218 AAEWDRLHPV) allowed for binding of KIR2DL2. Notably, functional analysis showed a significant inhibition of KIR2DL2(+) NK cells in the presence of p24 Gag209–218-pulsed T2 cells, while degranulation of KIR2DL2(−) NK cells was not affected. Moreover, we demonstrated that sequence variations in position 7 of this epitope observed frequently in naturally occurring HIV-1 sequences can modulate binding to KIR2DL2. Our results show that the majority of HIV-1 p24 Gag peptides stabilizing HLA-Cw*0102 do not allow for binding of KIR2DL2, but identified one HLA-Cw*0102-presented peptide (p24 Gag209–218) that was recognized by the inhibitory NK cell receptor KIR2DL2 leading to functional inhibition of KIR2DL2-expressing NK cells. Engagement of KIR2DL2 might protect virus-infected cells from NK cell-mediated lysis and selections of sequence polymorphisms that increase avidity to KIR2DL2 might provide a mechanism for HIV-1 to escape NK cell-mediated immune pressure.

No Evidence for Xenotropic Murine Leukemia-Related Virus Infection in Sweden Using Internally Controlled Multiepitope Suspension Array Serology

Many syndromes have a large number of differential diagnoses, a situation which calls for multiplex diagnostic systems. Myalgic encephalomyelitis (ME), also named chronic fatigue syndrome (CFS), is a common disease of unknown etiology. A mouse retrovirus, xenotropic murine leukemia-related virus (XMRV), was found in ME/CFS patients and blood donors, but this was not corroborated. However, the paucity of serological investigations on XMRV in humans prompted us to develop a serological assay which cover many aspects of XMRV antigenicity. It is a novel suspension array method, using a multiplex IgG assay with nine recombinant proteins from the env and gag genes of XMRV and 38 peptides based on known epitopes of vertebrate gammaretroviruses. IgG antibodies were sought in 520 blood donors and 85 ME/CFS patients and in positive- and negative-control sera from animals. We found no differences in seroreactivity between blood donors and ME/CFS patients for any of the antigens. This did not support an association between ME/CFS and XMRV infection. The multiplex serological system had several advantages: (i) biotinylated protein G allowed us to run both human and animal sera, which is essential because of a lack of XMRV-positive humans; (ii) a novel quality control was a pan-peptide positive-control rabbit serum; and (iii) synthetic XMRV Gag peptides with degenerate positions covering most of the variation of murine leukemia-like viruses did not give higher background than nondegenerate analogs. The principle may be used for creation of variant tolerant peptide serologies. Thus, our system allows rational large-scale serological assays with built-in quality control.

Characterization of HIV-Specific CD4+ T Cell Responses against Peptides Selected with Broad Population and Pathogen Coverage

CD4+ T cells orchestrate immunity against viral infections, but their importance in HIV infection remains controversial. Nevertheless, comprehensive studies have associated increase in breadth and functional characteristics of HIV-specific CD4+ T cells with decreased viral load. A major challenge for the identification of HIV-specific CD4+ T cells targeting broadly reactive epitopes in populations with diverse ethnic background stems from the vast genomic variation of HIV and the diversity of the host cellular immune system. Here, we describe a novel epitope selection strategy, PopCover, that aims to resolve this challenge, and identify a set of potential HLA class II-restricted HIV epitopes that in concert will provide optimal viral and host coverage. Using this selection strategy, we identified 64 putative epitopes (peptides) located in the Gag, Nef, Env, Pol and Tat protein regions of HIV. In total, 73% of the predicted peptides were found to induce HIV-specific CD4+ T cell responses. The Gag and Nef peptides induced most responses. The vast majority of the peptides (93%) had predicted restriction to the patient’s HLA alleles. Interestingly, the viral load in viremic patients was inversely correlated to the number of targeted Gag peptides. In addition, the predicted Gag peptides were found to induce broader polyfunctional CD4+ T cell responses compared to the commonly used Gag-p55 peptide pool. These results demonstrate the power of the PopCover method for the identification of broadly recognized HLA class II-restricted epitopes. All together, selection strategies, such as PopCover, might with success be used for the evaluation of antigen-specific CD4+ T cell responses and design of future vaccines.

Immune and Viral Correlates of “Secondary Viral Control” after Treatment Interruption in Chronically HIV-1 Infected Patients

Upon interruption of antiretroviral therapy, HIV-infected patients usually show viral load rebound to pre-treatment levels. Four patients, hereafter referred to as secondary controllers (SC), were identified who initiated therapy during chronic infection and, after stopping treatment, could control virus replication at undetectable levels for more than six months. In the present study we set out to unravel possible viral and immune parameters or mechanisms of this phenomenon by comparing secondary controllers with elite controllers and non-controllers, including patients under HAART. As candidate correlates of protection, virus growth kinetics, levels of intracellular viral markers, several aspects of HIV-specific CD4+ and CD8+ T cell function and HIV neutralizing antibodies were investigated. As expected all intracellular viral markers were lower in aviremic as compared to viremic subjects, but in addition both elite and secondary controllers had lower levels of viral unspliced RNA in PBMC as compared to patients on HAART. Ex vivo cultivation of the virus from CD4+ T cells of SC consistently failed in one patient and showed delayed kinetics in the three others. Formal in vitro replication studies of these three viruses showed low to absent growth in two cases and a virus with normal fitness in the third case. T cell responses toward HIV peptides, evaluated in IFN-γ ELISPOT, revealed no significant differences in breadth, magnitude or avidity between SC and all other patient groups. Neither was there a difference in polyfunctionality of CD4+ or CD8+ T cells, as evaluated with intracellular cytokine staining. However, secondary and elite controllers showed higher proliferative responses to Gag and Pol peptides. SC also showed the highest level of autologous neutralizing antibodies. These data suggest that higher T cell proliferative responses and lower replication kinetics might be instrumental in secondary viral control in the absence of treatment.

Immunodynamics of Th17 cells in HIV-1 subtype ‘C’ infection

Methods 31 HIV seropositive antiretroviral therapy naive and 8 HIV uninfected healthy control subjects were recruited and characterized as being early, late or slow progressor. Peripheral blood mononuclear cells were isolated from each study subject and stimulated with HIV-1 subtype ‘C’ gag peptide pool and assessed for IL-17 cytokine producing CD4-T cells using intracellular cytokine staining. All clinical groups were statistically compared by Kruskal-Wallis test and Spearman’s correlation coefficient was calculated for correlation of different variables.

The High Frequency of HIV Type 1-Specific Cellular Immune Responses in Seronegative Individuals with Parenteral and/or Heterosexual HIV Type 1 Exposure

Some individuals remain HIV seronegative despite repeated unprotected exposure to the virus. Recent observations led to a concept that acquired immunity plays a role in protection or at least in altered susceptibility to HIV-1 infection in highly exposed seronegative (ESN) individuals. Our aim was to study HIV-specific cellular immune responses induced in parenterally and/or heterosexually ESN individuals. Nine seronegative injection drug users (IDUs), 10 seronegative individuals, and nine of their HIV-positive sexual and/or IDU partners from the cohort of IDUs were included in the study. The discordant couples had unprotected sex, and some of seronegative partners also had parenteral exposure. Cell-mediated responses were measured in peripheral blood mononuclear cells (PBMCs) by ex vivo interferon (IFN)-γ-ELISpot and ICS combining IFN-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-2 after stimulation with four consensus peptide pools (Nef, Gag, RT, Env, subtype A-EE). Thirteen out of 19 (68%) seronegative study subjects had strong Nef peptide pool-specific ELISpot responses, three (16%) subjects responded against the Gag peptide pool, and one subject had an RT peptide pool response. Nef peptide pool responses in ESN were as high as in seropositive subjects. The multiple HIV-specific cytokine production in both CD4(+) and CD8(+) T cells was shown for several ESN subjects. The functional profiles of the immune responses were different between seronegative and HIV-positive study groups. Whether the observed cellular responses have any protective role against HIV needs to be further investigated.

STUDY ON THE EXPRESSION OF CO-STIMULATORY MARKER CD134 ON CD4+ T CELLS IN HIV-1–INFECTED INDIVIDUALS

CD4+CD25+CD134+ T cells play an important role in suppressing T cell responses to foreign pathogens, including human immunodeficiency virus (HIV). Thus, we aimed to investigate an increase in these populations in HIV-1–infected individuals. In this study, we used a panel of monoclonal antibodies staining of CD3/CD4/CD25/CD134. Without antigen stimulation, the expression of CD4+CD25+CD134+ T cells in 14 HIV-1–infected and 24 healthy individuals were 4.01% and 3.21%, respectively. However, there was an increase in the expression of CD4 + CD25+CD134+ T cells in HIV-1–infected individuals (6.85%) when stimulated with gag peptide. The upregulation of CD4+CD25+CD134+ T cells in HIV-1–infected individuals may result from activation of naturally occurring or by disease-associated antigen stimulation.

Human endogenous retrovirus K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected in HIV-1-infected subjects using standard peptide matrix-based screening.

T-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15 mer peptides. Small peptide pools and high concentrations were used to maximize sensitivity. In the 23 subjects studied, only three bona fide HERV-K(HML-2)-specific responses were detected. At these high peptide concentrations, we detected false-positive responses, three of which were mapped to an HIV-1 Gag peptide contaminant. Thus, HERV-K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected by 15 mer peptide mapping.

HIV-Specific CD4 T Cell Responses to Different Viral Proteins Have Discordant Associations with Viral Load and Clinical Outcome

A successful prophylactic vaccine is characterized by long-lived immunity, which is critically dependent on CD4 T cell-mediated helper signals. Indeed, most licensed vaccines induce antigen-specific CD4 T cell responses, in addition to high-affinity antibodies. However, despite the important role of CD4 T cells in vaccine design and natural infection, few studies have characterized HIV-specific CD4 T cells due to their preferential susceptibility to HIV infection. To establish at the population level the impact of HIV-specific CD4 T cells on viral control and define the specificity of HIV-specific CD4 T cell peptide targeting, we conducted a comprehensive analysis of these responses to the entire HIV proteome in 93 subjects at different stages of HIV infection. We show that HIV-specific CD4 T cell responses were detectable in 92% of individuals and that the breadth of these responses showed a significant inverse correlation with the viral load (P = 0.009, R = -0.31). In particular, CD4 T cell responses targeting Gag were robustly associated with lower levels of viremia (P = 0.0002, R = -0.45). Importantly, differences in the immunodominance profile of HIV-specific CD4 T cell responses distinguished HIV controllers from progressors. Furthermore, Gag/Env ratios were a potent marker of viral control, with a high frequency and magnitude of Gag responses and low proportion of Env responses associated with effective immune control. At the epitope level, targeting of three distinct Gag peptides was linked to spontaneous HIV control (P = 0.60 to 0.85). Inclusion of these immunogenic proteins and peptides in future HIV vaccines may act as a critical cornerstone for enhancing protective T cell responses.

Profile of T cell recognition of HIV type 1 consensus group M Gag and Nef peptides in a clade A1- and D-infected Ugandan population.

Reagents for evaluating non-clade B HIV-specific T cell responses are uncommon. Peptides based on highly conserved HIV-1 consensus group M sequences that are phylogenetically closer to most circulating strains may provide potential alternative reagents in populations with diverse infections, and may be relevant for vaccine design. Recognition of such reagents in clade A1-and D-infected populations has not been previously evaluated. Interferon (IFN)-γ ELISpot assay was used to evaluate T cell recognition of Gag and Nef peptides based on consensus group M sequences in 50 treatment-naive adults predominantly infected with HIV-1 clades A1 and D. Gag-induced T cell responses were correlated with gag sequence diversity. Infecting clades were determined from gag sequences for 45 of the 50 subjects as 40% clade A1 (18/45), 45% clade D (20/45), 2% clade C (1/45), 2% A1/C recombinant (1/45), 2% A1/D (1/45), 7% CRF10_CD (3/45), and 2% U (unclassifiable) (1/45). The mean genetic divergence and diversity of clade A and D gag region compared to group M consensus sequences at synonymous and nonsynonymous nucleotide and amino acid levels were not always significant. Gag peptides were targeted at significantly higher frequency [88% (44/50)] than Nef [64% (32/50)]; p=0.014, although their mean IFN-γ magnitudes were comparable ([3703 (95% CI 2567-4839)] vs. [2120 (95% CI 478-3762)]), respectively. Measurable virus-induced IFN-γ responses were detected in 96% (48/50) individuals, primarily targeting the more conserved Gag p24 and Nef central core regions. Use of these reagents to screen for HIV-specific IFN-γ responses may mitigate the challenge of viral diversity; although this targeting is apparently biased toward a few highly conserved epitopes.

Mice chronically infected with chimeric HIV resist peripheral and brain superinfection: a model of protective immunity to HIV.

Infection by some viruses induces immunity to reinfection, providing a means to identify protective epitopes. To investigate resistance to reinfection in an animal model of HIV disease and its control, we employed infection of mice with chimeric HIV, EcoHIV. When immunocompetent mice were infected by intraperitoneal (IP) injection of EcoHIV, they resisted subsequent secondary infection by IP injection, consistent with a systemic antiviral immune response. To investigate the potential role of these responses in restricting neurotropic HIV infection, we established a protocol for efficient EcoHIV expression in the brain following intracranial (IC) inoculation of virus. When mice were inoculated by IP injection and secondarily by IC injection, they also controlled EcoHIV replication in the brain. To investigate their role in EcoHIV antiviral responses, CD8+ T lymphocytes were isolated from spleens of EcoHIV infected and uninfected mice and adoptively transferred to isogenic recipients. Recipients of EcoHIV primed CD8+ cells resisted subsequent EcoHIV infection compared to recipients of cells from uninfected donors. CD8+ spleen cells from EcoHIV-infected mice also mounted modest but significant interferon-γ responses to two HIV Gag peptide pools. These findings suggest EcoHIV-infected mice may serve as a useful system to investigate the induction of anti-HIV protective immunity for eventual translation to human beings.