Gag-specific Responses Research Articles

LAMP-1 Chimeric to HIV-1 p55Gag in the Immunization of Neonate Mice Induces an Early Germinal Center Formation and AID Expression.

Neonates have a limited adaptive response of plasma cells, germinal center (GC) B cells, and T follicular helper cells (TFH). As neonatal vaccination can be an important tool for AIDS prevention, these limitations need to be overcome. Chimeric DNA vaccine encoding p55Gag HIV-1 protein conjugated with lysosomal-associated membrane protein 1 (LAMP-1) has been described as immunogenic in the neonate period. Herein, we investigated the immunologic mechanisms involved in neonatal immunization with a LAMP-1/p55Gag (LAMP/Gag) DNA vaccine in a C57BL/6 mouse background. Neonatal LAMP/Gag vaccination induced strong Gag-specific T-cell response until adulthood and elevated levels of anti-Gag IgG antibodies. We also demonstrated for the first time that the immunogenicity of the neonatal period with LAMP/Gag is due to the induction of high-affinity anti-p24 IgG antibodies and long-term plasma cells. Together with that, there is the generation of early TFH cells and the formation of GC sites with the upregulation of activation-induced cytidine deaminase (AID) enzyme mRNA and protein expression in draining lymph nodes after neonatal LAMP/Gag vaccination. These findings underscore that the LAMP-1 strategy in the chimeric vaccine could be useful to enhance antibody production even in the face of neonatal immaturity, and they contribute to the development of new vaccine approaches for other emerging pathogens at an early stage of life.

Highly dampened HIV-specific cytolytic effector T cell responses define viremic non-progression

This study reports on HIV-specific T cell responses in HIV-1 infected Viremic Non-Progressors (VNPs), a rare group of people living with HIV that exhibit asymptomatic infection over several years accompanied by stable CD4+ T cell counts in spite of ongoing viral replication. We attempted to identify key virus-specific functional attributes that could underlie the apparently paradoxical virus-host equilibrium observed in VNPs. Our results revealed modulation of HIV-specific CD4+ and CD8+ effector T cell responses in VNPs towards a dominant non-cytolytic profile with concomitantly diminished degranulation (CD107a+) ability. Further, the HIV specific CD8+ effector T cell response was primarily enriched for MIP-1β producing cells. As expected, concordant with better viral suppression, VCs exhibit a robust cytolytic T cell response. Interestingly, PuPs shared features common to both these responses but did not exhibit a CD4+ central memory IFN-γ producing Gag-specific response that was shared by both non-progressor (VC and VNP) groups, suggesting CD4 helper response is critical for non-progression. Our study also revealed that cytolytic response in VNPs is primarily limited to polyfunctional cells while both monofunctional and polyfunctional cells significantly contribute to cytolytic responses in VCs. To further understand mechanisms underlying the unique HIV-specific effector T cell response described here in VNPs we also evaluated and demonstrated a possible role for altered gut homing in these individuals. Our findings inform immunotherapeutic interventions to achieve functional cures in the context of ART resistance and serious non AIDS events.

A longitudinal analysis of immune escapes from HLA-B*13-restricted T-cell responses at early stage of CRF01_AE subtype HIV-1 infection and implications for vaccine design

BackgroundIdentifying immunogens which can elicit effective T cell responses against human immunodeficiency virus type 1 (HIV-1) is important for developing a T-cell based vaccine. It has been reported that human leukocyte antigen (HLA)-B*13-restricted T-cell responses contributed to HIV control in subtype B′ and C infected individuals. However, the kinetics of B*13-restricted T-cell responses, viral evolution within epitopes, and the impact on disease progression in CRF01_AE subtype HIV-1-infected men who have sex with men (MSM) are not known.ResultsInterferon-γ ELISPOT assays and deep sequencing of viral RNAs were done in 14 early HLA-B*13-positive CRF01_AE subtype HIV-1-infected MSM. We found that responses to RQEILDLWV (Nef106–114, RV9), GQMREPRGSDI (Gag226–236, GI11), GQDQWTYQI (Pol487–498, GI9), and VQNAQGQMV (Gag135–143, VV9) were dominant. A higher relative magnitude of Gag-specific T-cell responses, contributed to viral control, whereas Nef-specific T-cell responses were associated with rapid disease progression. GI11 (Gag) was conserved and strong GI11 (Gag)-specific T-cell responses showed cross-reactivity with a dominant variant, M228I, found in 3/12 patients; GI11 (Gag)-specific T-cell responses were positively associated with CD4 T-cell counts (R = 0.716, P = 0.046). Interestingly, the GI9 (Pol) epitope was also conserved, but GI9 (Pol)-specific T-cell responses did not influence disease progression (P > 0.05), while a D490G variant identified in one patient did not affect CD4 T-cell counts. All the other epitopes studied [VV9 (Gag), RQYDQILIEI (Pol113–122, RI10), HQSLSPRTL (Gag144–152, HL9), and RQANFLGRL (Gag429–437, RL9)] developed escape mutations within 1 year of infection, which may have contributed to overall disease progression. Intriguingly, we found early RV9 (Nef)-specific T-cell responses were associated with rapid disease progression, likely due to escape mutations.ConclusionsOur study strongly suggested the inclusion of GI11 (Gag) and exclusion of RV9 (Nef) for T-cell-based vaccine design for B*13-positive CRF01_AE subtype HIV-1-infected MSM and high-risk individuals.

Diminished Peripheral CD29hi Cytotoxic CD4+ T Cells Are Associated With Deleterious Effects During SIV Infection.

Cytotoxic CD4+ T cells (CD4+ CTLs) limit HIV pathogenesis, as evidenced in elite controllers (a subset of individuals who suppress the virus without the need for therapy). CD4+ CTLs have also been shown to kill HIV-infected macrophages. However, little is known about their contribution towards HIV persistence, how they are affected following exposure to immune modulators like morphine, and what factors maintain their frequencies and function. Further, the lack of robust markers to identify CD4+ CTLs in various animal models limits understanding of their role in HIV pathogenesis. We utilized various PBMC samples obtained from SIV infected and cART treated rhesus macaques exposed to morphine or saline and subjected to flow cytometry evaluations. Thereafter, we compared and correlated the expression of CD4+ CTL-specific markers to viral load and viral reservoir estimations in total CD4+ T cells. We found that CD29 could be reliably used as a marker to identify CD4+ CTLs in rhesus macaques since CD29hi CD4+ T cells secrete higher cytotoxic and proinflammatory cytokines following PMA/ionomycin or gag stimulation. In addition, this immune cell subset was depleted during untreated SIV infection. Strikingly, we also observed that early initiation of cART reconstitutes depleted CD29hi CD4+ T cells and restores their function. Furthermore, we noted that morphine exposure reduced the secretion of proinflammatory cytokines/cytotoxic molecules in CD29hi CD4+ T cells. Lastly, increased functionality of CD29hi CD4+ T cells as depicted by elevated levels of either IL-21 or granzyme B hi T Bet+ gag specific responses were linked to limiting the size of the replication-competent reservoir during cART treatment. Collectively, our data suggest that CD4+ CTLs are crucial in limiting SIV pathogenesis and persistence.

Reversal of Viral Latency and Induction of Gag-Specific T-Cell Responses in HIV-1-Infected Adults Through Cyclic Treatment Interruption of Rosuvastatin: A Proof-of-Concept Study.

HIV-1 infection remains incurable through combination antiretroviral therapy. Previous studies have shown statins have immunomodulatory effects, and interruption of statins may cause an immune rebound. In this proof-of-concept study, we longitudinally assessed the impact of immune rebound by cyclic treatment-interruption (CTI) of rosuvastatin on the reversal of HIV latency. The HIV-1-infected persons with stable viral control were considered to be enrolled for CTI of rosuvastatin with a fixed 12-week interval for 72 weeks (3 treatment-interruption cycles). HIV-1 Gag-specific T-cell responses, cell-associated RNA, and proviral DNA were determined. From Feb 2017 to Dec 2019, 10 subjects were enrolled. During the 72-week follow-up, their CD4+ T-cell counts did not significantly change, and plasma HIV RNA remained undetectable. Transient but remarkable increases in levels of cell-associated RNA, Gag-specific interferon-γ production from CD4+ T cells and Gag-specific CD8+ cytotoxic capacity were detected shortly after stopping rosuvastatin in every cycle of CTI of rosuvastatin. Furthermore, there was a 2.63-fold reduction (range, 1.41-4.82) in proviral DNA levels (P = 0.005) during the 72-week follow-up. A significant linear association was demonstrated between their nadir CD4+ T-cell counts and the fold decrease in proviral DNA levels (R = 0.81, P = 0.004). It may be possible to reverse viral latency in CD4+ T cells, activate Gag-specific T cells, and reduce viral reservoir size through CTI of rosuvastatin in HIV-1-infected subjects with stable combination antiretroviral therapy, especially in those with nadir CD4+ T-cell counts > 350 cells/μL.

Modified Adenovirus Prime-Protein Boost Clade C HIV Vaccine Strategy Results in Reduced Viral DNA in Blood and Tissues Following Tier 2 SHIV Challenge.

Designing immunogens and improving delivery methods eliciting protective immunity is a paramount goal of HIV vaccine development. A comparative vaccine challenge study was performed in rhesus macaques using clade C HIV Envelope (Env) and SIV Gag antigens. One group was vaccinated using co-immunization with DNA Gag and Env expression plasmids cloned from a single timepoint and trimeric Env gp140 glycoprotein from one of these clones (DNA+Protein). The other group was a prime-boost regimen composed of two replicating simian (SAd7) adenovirus-vectored vaccines expressing Gag and one Env clone from the same timepoint as the DNA+Protein group paired with the same Env gp140 trimer (SAd7+Protein). The env genes were isolated from a single pre-peak neutralization timepoint approximately 1 year post infection in CAP257, an individual with a high degree of neutralization breadth. Both DNA+Protein and SAd7+Protein vaccine strategies elicited significant Env-specific T cell responses, lesser Gag-specific responses, and moderate frequencies of Env-specific TFH cells. Both vaccine modalities readily elicited systemic and mucosal Env-specific IgG but not IgA. There was a higher frequency and magnitude of ADCC activity in the SAd7+Protein than the DNA+Protein arm. All macaques developed moderate Tier 1 heterologous neutralizing antibodies, while neutralization of Tier 1B or Tier 2 viruses was sporadic and found primarily in macaques in the SAd7+Protein group. Neither vaccine approach provided significant protection from viral acquisition against repeated titered mucosal challenges with a heterologous Tier 2 clade C SHIV. However, lymphoid and gut tissues collected at necropsy showed that animals in both vaccine groups each had significantly lower copies of viral DNA in individual tissues compared to levels in controls. In the SAd7+Protein-vaccinated macaques, total and peak PBMC viral DNA were significantly lower compared with controls. Taken together, this heterologous Tier 2 SHIV challenge study shows that combination vaccination with SAd7+Protein was superior to combination DNA+Protein in reducing viral seeding in tissues in the absence of protection from infection, thus emphasizing the priming role of replication-competent SAd7 vector. Despite the absence of correlates of protection, because antibody responses were significantly higher in this vaccine group, we hypothesize that vaccine-elicited antibodies contribute to limiting tissue viral seeding.

Conserved broad HIV-1 Gag-specific responses associated with low viral load and high CD4+ T cell nadir and preserved HAART regimen.

Broad human immunodeficiency virus type 1 (HIV-1) Gag-specific cellular responses can control viremia and provide slow progression to Acquired immunodeficiency syndrome (AIDS). In this study, we evaluate multiple HIV-1 Gag-specific lymphoproliferative responses and find their connection with cluster of differentiation 4 (CD4)+ T cell count and viral load from chronically HIV-1-infected patients. We further search for the correlation between multiple Gag-specific lymphoproliferative responses and changes in highly active antiretroviral therapy (HAART) regimen. We found correlation between Gag-specific responses and higher CD4+ T cells nadir and low HIV-1 viral load. Additionally, we observed that HIV-1-infected subjects did not need to change HAART regimen, when multiple Gag responses are present. We concluded that the start of HAART when CD4+ T cell nadir is the highest as possible may promote Gag-specific cellular responses conservation. Multiple Gag responses must be important to suppress HIV-1 replication. Preserved Gag-specific responses reduce HIV-1 viral load and are associated with stability of HAART regimen. Keywords: HIV-1; Gag; lymphoproliferation; viral load; HAART.

Abstract 5531: K0401-020 anti-VISTA antibody monotherapy increases specific CD8 T cell response in non-human primates

Abstract Background: VISTA is a B7 family protein described as a negative checkpoint of T cell responses, both in autoimmunity and cancer murine models. K01401-020 is a novel IND enabling anti-VISTA antibody. In vitro, when incubated with human blood cells, K01401-020 stimulates NK cells, monocytes and cytokine production, contributing to T cell activation. In vivo, in non-human primates, K01401-020 notably induced dendritic cell activation (Loukili et al, AACR 2019). However, so far, no in vivo demonstration of anti-VISTA pharmacodynamic effect on T cells has been demonstrated beyond mouse models. Thus, we explored the effect of anti-VISTA antibody on CD8 T cell specific activation in non-human primates, using SIV immunization as a model. Methods: The detection of T cell response amplification by immune-checkpoint inhibitors is very challenging in primates during standard PK or safety studies in the absence of adequate antigen stimulation. KLH is described as an immunogen in monkeys but induces mainly a humoral (IgG/IgM) and not a CD8 response, and thus cannot reveal the pharmacodynamic activity of immune-checkpoint inhibitors. Here, we explored well-described model of primate immunization that induces specifically a cellular response including CD8 (Martinon et al, Hum Gene Ther. 2009) to reveal the activity of Immune-checkpoints inhibitors. Groups of three adult cynomolgus macaques were immunized thrice with 1mg of MultiSIVmac239 GTU-DNA, by intradermal route with electroporation. One-day after the last immunization, animals received a single intravenous injection of either vehicle, anti-PD1 antibody (pembrolizumab) or anti-VISTA antibody K01401-020. IFNγ ELISPOT assay was performed on PBMCs in the presence of either Nef RM9 peptide or Gag (peptide pool), while RM9 tetramer assay and cell subsets phenotyping were performed on whole blood. Results: RM9 and gag-specific CD8 responses were evidenced as soon as after the second immunization in at least 2/3 animals from all groups. The third immunization reactivated CD8-specific responses in all groups. Pembrolizumab and anti-VISTA antibody clearly amplified CD8 specific responses respectively in 2/3 and 3/3 monkeys. Conclusions: Here we show for the first time that a single anti-VISTA antibody infusion monotherapy in non-human primates potentializes specific CD8 T cell responses against exogenous antigen. The above data suggests that anti-VISTA antibody K01401-020 given as a single agent may effectively boosts CD8 T cell activity against tumor antigens. Confidential – Pierre Fabre and CEA collaboration Citation Format: Christine Libon, Isabelle Vandenberghe, Romain Marlin, Wesley Gros, Marco Leonec, Mario Gomez-Pacheco, Anne-Sophie Gallouet, Françoise Fraboul, Abderrahim Mahfoudi, Nathalie Dereuddre-Bosquet, Pierre Ferré. K0401-020 anti-VISTA antibody monotherapy increases specific CD8 T cell response in non-human primates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5531.

T cells with high PD-1 expression are associated with lower HIV-specific immune responses despite long-term antiretroviral therapy.

We evaluated frequencies of T cells with high PD-1 expression (PD-1) before and after long-term effective antiretroviral therapy (ART), and determined if frequencies on-ART correlated positively with measures of HIV persistence and negatively with HIV-specific responses. We enrolled individuals who started ART during chronic infection and had durable suppression of viremia for at least 4 years (N = 99). We assessed PD-1 T-cell frequencies at timepoints pre-ART and on-ART using flow cytometry, and evaluated how frequencies on-ART are associated with measures of HIV persistence, HIV-specific immune responses, and immune activation levels. Pre-ART, PD-1 CD4 T cells correlated positively with viremia and negatively with CD4 T-cell count. At year 1 on-ART, %PD-1 CD4 T cells decreased but then remained stable at 4 and 6-15 years on-ART, whereas %PD-1 CD8 T cells on-ART remained similar to pre-ART. PD-1 CD4 T cells correlated positively with HIV DNA pre-ART and on-ART, and with CD4 T-cell activation on-ART. PD-1 CD4 T cells negatively correlated with HIV Gag-specific and Env-specific T-cell responses but not with CMV-specific or EBV-specific responses. PD-1 CD8 T cells trended towards a negative correlation with responses to Gag and Env, but not to CMV and EBV. PD-1 T cells persist in blood despite prolonged suppression on ART, correlate with HIV DNA levels, and are associated with lower HIV-specific T-cell responses but not CMV-specific or EBV-specific responses, suggesting that these cells are HIV-specific. The findings support evaluating PD-1 blockade strategies for their effect on HIV persistence and HIV-specific immunity.

DNA prime/MVTT boost regimen with HIV-1 mosaic Gag enhances the potency of antigen-specific immune responses

HIV-1 diversity and latent reservoir are the major challenges for the development of an effective AIDS vaccine. It is well indicated that Gag-specific CD8+ T cells serve as the dominant host immune surveillance for HIV-1 control, but it still remains a challenge for vaccine design to induce broader and stronger cytotoxic T cell immunity against the virus. Genetic variation of the HIV-1 gag gene across different clades is one of the reasons for the reduction of antigenic epitope coverage. Here, we report an immunization strategy with heterologous vaccines expressing a mosaic Gag antigen aimed to increase antigenic breadth against a wider spectrum of HIV-1 strains. Priming using a DNA vaccine via in vivo electroporation, followed by boosting with a live replication-competent modified vaccinia TianTan (MVTT) vectored vaccine, elicited greater and broader protective Gag-specific immune responses in mice. Compared to DNA or MVTT homologous immunization, the heterologous DNA/MVTT vaccination resulted in higher frequencies of broadly reactive, Gag-specific, polyfunctional, long-lived cytotoxic CD8+ T cells, as well as increased anti-Gag antibody titer. Importantly, the DNA/MVTT heterologous vaccination induced protection against EcoHIV and mesothelioma AB1-Gag challenges. In summary, the stronger protective Gag-specific immunity induced by the heterologous regimen using two safe vectors shows promise for further development to enhance anti-HIV-1 immunity. Our study has important implications for immunogen design and the development of an effective HIV-1 heterologous vaccination strategy.

Viral control in chronic HIV-1 subtype C infection is associated with enrichment of p24 IgG1 with Fc effector activity.

Postinfection HIV viral control and immune correlates analysis of the RV144 vaccine trial indicate a potentially critical role for Fc receptor-mediated antibody functions. However, the influence of functional antibodies in clade C infection is largely unknown. Plasma samples from 361 chronic subtype C-infected, antiretroviral therapy-naive participants were tested for their HIV-specific isotype and subclass distributions, along with their Fc receptor-mediated functional potential. Total IgG, IgG subclasses and IgA binding to p24 clade B/C and gp120 consensus C proteins were assayed by multiplex. Antibody-dependent uptake of antigen-coated beads and Fc receptor-mediated natural killer cell degranulation were evaluated as surrogates for antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), respectively. p24 IgG1 was the only subclass associated with viral control (P = 0.01), with higher p24-specific ADCP and ADCC responses detected in individuals with high p24 IgG1. Although p24 IgG1 levels were enriched in patients with elevated Gag-specific T-cell responses, these levels remained an independent predictor of low-viral loads (P = 0.04) and high CD4+ cell counts (P = 0.004) after adjusting for Gag-specific T-cell responses and for protective HLA class I alleles. p24 IgG1 levels independently predict viral control in HIV-1 clade C infection. Whether these responses contribute to direct antiviral control via the recruited killing of infected cells via the innate immune system or simply mark a qualitatively superior immune response to HIV, is uncertain, but highlights the role of p24-specific antibodies in control of clade C HIV-1 infection.

Control of HIV-1 Pathogenesis in Viremic Nonprogressors Is Independent of Gag-Specific Cytotoxic T Lymphocyte Responses.

ABSTRACTViremic nonprogressors (VNPs) constitute a very scarce group of untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain stable CD4+ T cell counts despite high levels of HIV-1 replication. The specific factors associated with this atypical control of the HIV infection have been poorly described. Since specific T cell responses seem to be one of the main causes of HIV-1 control in elite controllers, we studied whether HIV-1 Gag-specific cytotoxic T lymphocyte (CTL) responses could also modulate disease control in VNPs. We characterized the immune responses from four VNPs compared to those of five standard progressors (SPs) during the first years of HIV-1 infection. We observed no differences in the breadth and frequency of Gag-specific cellular responses. Furthermore, we obtained 217 HIV-1Gag clonal sequences in which the viral variability of Gag increased over 3 years of infection for synonymous and nonsynonymous mutations in both VNPs and SPs. VNPs evolution rates in gag were comparable to SPs. This observation is in line with a similar accumulation of CTL putative escape mutations in Gag epitopes targeted by CTL responses. Altogether, the absence of viral pathogenesis in VNP individuals seems to be independent of HIV-Gag-specific CTL responses. This novel information guides to the study of alternative mechanism of HIV-1 pathogenesis control.IMPORTANCE Control of HIV infection has been widely studied in elite controllers or long-term nonprogressor models. However, there is a less-known group of individuals, termed viremic nonprogressors (VNPs), who maintain stable CD4+ T cell counts despite high plasma viremia. The mechanisms involved in this remarkable control of HIV-1 pathogenesis clearly have implications for the development of new drugs and vaccines. We show here for the first time that VNPs have immune responses and HIV-gag evolution similar to those of standard progressors. Remarkably, we demonstrate that the mechanism of pathogenesis control in these individuals differs from some elite controllers that are reported to have improved immune control. This is noteworthy since it opens the door to new, as-yet-unknown mechanisms for HIV control. Our novel results advance the understanding of mechanisms involved in viremic nonprogression and suggest that there are alternative mechanisms to the adaptive immune responses for an effective control of viral pathogenesis.

Therapeutic conserved elements (CE) DNA vaccine induces strong T-cell responses against highly conserved viral sequences during simian-human immunodeficiency virus infection

ABSTRACTHIV-specific T-cell responses play a key role in controlling HIV infection, and therapeutic vaccines for HIV that aim to improve viral control will likely need to improve on the T-cell responses induced by infection. However, in the setting of chronic infection, an effective therapeutic vaccine must overcome the enormous viral genetic diversity and the presence of pre-existing T-cell responses that are biased toward immunodominant T-cell epitopes that can readily mutate to evade host immunity and thus potentially provide inferior protection. To address these issues, we investigated a novel, epidermally administered DNA vaccine expressing SIV capsid (p27Gag) homologues of highly conserved elements (CE) of the HIV proteome in macaques experiencing chronic but controlled SHIV infection. We assessed the ability to boost or induce de novo T-cell responses against the conserved but immunologically subdominant CE epitopes. Two groups of animals were immunized with either the CE DNA vaccine or a full-length SIV p57gag DNA vaccine. Prior to vaccination, CE responses were similar in both groups. The full-length p57gag DNA vaccine, which contains the CE, increased overall Gag-specific responses but did not increase CE responses in any animals (0/4). In contrast, the CE DNA vaccine increased CE responses in all (4/4) vaccinated macaques. In SIV infected but unvaccinated macaques, those that developed stronger CE-specific responses during acute infection exhibited lower viral loads. We conclude that CE DNA vaccination can re-direct the immunodominance hierarchy towards CE in the setting of attenuated chronic infection and that induction of these responses by therapeutic vaccination may improve immune control of HIV.

Factors Leading to the Loss of Natural Elite Control of HIV-1 Infection.

HIV-1 elite controllers (EC) maintain undetectable viral loads (VL) in the absence of antiretroviral treatment. However, these subjects have heterogeneous clinical outcomes, including a proportion that loses HIV-1 control over time. In this work, we compared, in a longitudinal design, transient EC, analyzed before and after the loss of virological control, with persistent EC. The aim was to identify factors leading to the loss of natural virological control of HIV-1 infection with a longitudinal retrospective study design. Gag-specific T-cell responses were assessed by in vitro intracellular polycytokine production quantified by flow cytometry. Viral diversity determinations and sequence dating were performed in proviral DNA by PCR amplification at limiting dilution of env and gag genes. The expression profile of 70 serum cytokines and chemokines was assessed by multiplex immunoassays. We identified transient EC as subjects with low Gag-specific T-cell polyfunctionality, high viral diversity, and high proinflammatory cytokine levels before the loss of control. Gag-specific T-cell polyfunctionality was inversely associated with viral diversity in transient controllers before the loss of control (r = -0.8; P = 0.02). RANTES was a potential biomarker of transient control. This study identified virological and immunological factors, including inflammatory biomarkers associated with two different phenotypes within EC. These results may allow a more accurate definition of EC, which could help in better clinical management of these individuals and in the development of future curative approaches.IMPORTANCE There is a rare group of HIV-infected patients who have the extraordinary capacity to maintain undetectable viral load levels in the absence of antiretroviral treatment, the so-called HIV-1 elite controllers (EC). However, there is a proportion within these subjects that eventually loses this capability. In this work, we found differences in virological and immune factors, including soluble inflammatory biomarkers, between subjects with persistent control of viral replication and EC that will lose virological control. The identification of these factors could be a key point for a right medical care of those EC who are going to lose natural control of viral replication and for the design of future immunotherapeutic strategies using as a model the natural persistent control of HIV infection.

HIV-1 Gag specific IgG response in mice immunized with Vp22-Gag vaccine candidate

Latar Belakang: Stimulasi respon sel T CD8+ spesifik Gag, terkait dengan penurunan viremia, kontrol replikasi virus, dan perkembangan penyakit yang lambat. Respon T CD8+ yang efektif juga dipengaruhi oleh sel T CD4+. Protein rekombinan Gag dapat diklona dan diekpresikan pada sistem prokariota, dan pada saat diimunisasi pada hewan coba atau manusia akan bersifat sebagai antigen eksogen. Antigen eksogen dapat menjadi antigen endogen dengan menambahkan protein yang mempunyai kemampuan bertranslokasi kedalam membran sel, salah satunya protein Vp22.Metode: Transformasi Plasmid rekombinan didapatkan dari Pusat Penelitian dan Layanan Virologi Kanker Patobiologi Fakultas Kedokteran Universitas Indonesia-Rumah Sakit Pusat Nasional dr. Cipto Mangunkusumo (PPLVKP FK UI-RSCM), yang dilakukan transformasi pada sistem ekpresi prokariota dengan metode heat shock, yang dilanjutkan dengan ekpresi protein rekombinan. Purifikasi protein rekombinan dilakukan dengan kromatografi afinitas. Analisa berat molekul protein rekombinan dilakukan dengan SDS-PAGE. Western blotting dilakukan untuk mengaetahui reaktifitas protein rekombinan dengan antibodi poliklonal terhadap antigen p24. Transfeksi sel CHO dan imunisasi mencit DDY dengan protein rekombinan, untuk mengetahui kemampuan migrasi intraseluler serta stimulasi respon imun spesifik.Hasil: Uji western blotting, menunjukkan protein rekombinan dapat berinteraksi dengan antibodi poliklonal terhadap antigen p24. Pengamatan mikroskop konfokal menunjukkan protein rekombinan berlokalisasi dengan endosom. Uji ELISA, menunjukkan respon IgG spesifik Gag setelah imunisasi pada mencit DDY. Kesimpulan: Protein rekombinan dapat diekspresikan pada sistem ekspresi prokariota. Kemampuan migrasi intrasseluler protein rekombinan pada sel CHO belum dapat dibuktikan. Protein rekombinan dapat menstimulasi respon IgG spesifik Gag. Kata Kunci: Protein rekombinan Gag dan Vp22-Gag; Migrasi intraseluler, Respon IgG spesifik Gag. Abstract Background: Stimulation of Gag-specific CD8+ T-cell response, associated with reduction in viremia, viral replication control, and slow disease progression. Effective CD8+ T cell response is also influenced by CD4+ T cells. Gag recombinant protein may be cloned and expressed in the prokaryotic system and when they are immunized in experimental animals or human will have property as exogenous antigens. Exogenous antigens may become endogenous antigens by adding proteins that have the ability to translocate into the cell membrane, one of which is the Vp22 protein. Methods: Recombinant plasmids were obtained from Research and Services Centers of Virology and Cancer Patobiology Medical Faculity Universitas Indonesia-dr. Cipto Mangunkusumo National Central General Hospital (PPLVKP FK UI-RSCM) which transformation to prokaryotic expression system with heat shock method was followed by expression of recombinant proteins. Purification of recombinant proteins was performed with affinity chromatography. The molecular weight analysis of recombinant proteins was performed with SDS-PAGE. Western blotting was performed to determine the reactivity of recombinant proteins with polyclonal antibodies against p24 antigens. Transfection of Chinese Hamster Ovary (CHO) cell and immunization of Deutschland, Denken, and Yoken (DDY) mice with recombinant proteins was conducted to determine intracellular migration ability and stimulation of specific immune response. Results: Western blotting test, indicating recombinant protein may interact with polyclonal antibody against p24 antigens. The observation of a confocal microscope showed recombinant proteins localized with endosomes. The Enzyme-linked Immunosorbent Assay (ELISA) test indicates Gag-specific IgG response after immunization in DDY mice.Conclusion: Recombinant proteins may be expressed on a prokaryotic expression system. The ability of recombinant protein intracellular migration in CHO cell has not been proven. Recombinant proteins may stimulate Gag-specific IgG response. Keywords: Gag and Vp22-Gag recombinant proteins; intracellular migration, Gag-specific IgG response.

Vector Order Determines Protection against Pathogenic Simian Immunodeficiency Virus Infection in a Triple-Component Vaccine by Balancing CD4+ and CD8+ T-Cell Responses.

An effective AIDS vaccine should elicit strong humoral and cellular immune responses while maintaining low levels of CD4+ T-cell activation to avoid the generation of target cells for viral infection. The present study investigated two prime-boost regimens, both starting vaccination with single-cycle immunodeficiency virus, followed by two mucosal boosts with either recombinant adenovirus (rAd) or fowlpox virus (rFWPV) expressing SIVmac239 or SIVmac251 gag/pol and env genes, respectively. Finally, vectors were switched and systemically administered to the reciprocal group of animals. Only mucosal rFWPV immunizations followed by systemic rAd boost significantly protected animals against a repeated low-dose intrarectal challenge with pathogenic SIVmac251, resulting in a vaccine efficacy (i.e., risk reduction per exposure) of 68%. Delayed viral acquisition was associated with higher levels of activated CD8+ T cells and Gag-specific gamma interferon (IFN-γ)-secreting CD8+ cells, low virus-specific CD4+ T-cell responses, and low Env antibody titers. In contrast, the systemic rFWPV boost induced strong virus-specific CD4+ T-cell activity. rAd and rFWPV also induced differential patterns of the innate immune responses, thereby possibly shaping the specific immunity. Plasma CXCL10 levels after final immunization correlated directly with virus-specific CD4+ T-cell responses and inversely with the number of exposures to infection. Also, the percentage of activated CD69+ CD8+ T cells correlated with the number of exposures to infection. Differential stimulation of the immune response likely provided the basis for the diverging levels of protection afforded by the vaccine regimen.IMPORTANCE A failed phase II AIDS vaccine trial led to the hypothesis that CD4+ T-cell activation can abrogate any potentially protective effects delivered by vaccination or promote acquisition of the virus because CD4+ T helper cells, required for an effective immune response, also represent the target cells for viral infection. We compared two vaccination protocols that elicited similar levels of Gag-specific immune responses in rhesus macaques. Only the animal group that had a low level of virus-specific CD4+ T cells in combination with high levels of activated CD8+ T cells was significantly protected from infection. Notably, protection was achieved despite the lack of appreciable Env antibody titers. Moreover, we show that both the vector and the route of immunization affected the level of CD4+ T-cell responses. Thus, mucosal immunization with FWPV-based vaccines should be considered a potent prime in prime-boost vaccination protocols.

Host genotype and time dependent antigen presentation of viral peptides: predictions from theory

The rate of progression of HIV infected individuals to AIDS is known to vary with the genotype of the host, and is linked to their allele of human leukocyte antigen (HLA) proteins, which present protein degradation products at the cell surface to circulating T-cells. HLA alleles are associated with Gag-specific T-cell responses that are protective against progression of the disease. While Pol is the most conserved HIV sequence, its association with immune control is not as strong. To gain a more thorough quantitative understanding of the factors that contribute to immunodominance, we have constructed a model of the recognition of HIV infection by the MHC class I pathway. Our model predicts surface presentation of HIV peptides over time, demonstrates the importance of viral protein kinetics, and provides evidence of the importance of Gag peptides in the long-term control of HIV infection. Furthermore, short-term dynamics are also predicted, with simulation of virion-derived peptides suggesting that efficient processing of Gag can lead to a 50% probability of presentation within 3 hours post-infection, as observed experimentally. In conjunction with epitope prediction algorithms, this modelling approach could be used to refine experimental targets for potential T-cell vaccines, both for HIV and other viruses.

Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen

Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gagp37 and two vaccinations with MVA-CMDR encoding subtype A Gagp55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ+) Gag-specific T-cell responses were dominated by CD4+ T cells (P < 0.001 compared to CD8+ T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gagp24 regions, more conserved between prime and boost, compared to those of regions within Gagp15 (not primed) and Gagp17 (less conserved; P < 0.0001 for both). Four regions within Gagp24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens (P = 0.04, r2 = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4+ T cells and that targeted multiple antigenic regions within the conserved Gagp24 protein.IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a given immunogen in prime-boost vaccination strategies is one approach that aims to improve coverage for global virus variants, the immunologic consequences of this strategy have been poorly defined so far. It is unclear whether inclusion of multiple variants in prime-boost vaccination strategies improves recognition of variant viruses by T cells and by which mechanisms this would be achieved, either by improved cross-recognition of multiple variants for a given antigenic region or through preferential targeting of antigenic regions more conserved between prime and boost. Engineering vaccines to induce adaptive immune responses that preferentially target conserved antigenic regions of viral vulnerability might facilitate better immune control after preventive and therapeutic vaccination for HIV and for other variable viruses.

A Randomized Controlled Trial of Lisinopril to Decrease Lymphoid Fibrosis in Antiretroviral-Treated, HIV-infected Individuals.

Background:In HIV infection, lymphoid tissue is disrupted by fibrosis. Angiotensin converting enzyme inhibitors have anti-fibrotic properties. We completed a pilot study to assess whether the addition of lisinopril to antiretroviral therapy (ART) reverses fibrosis of gut tissue, and whether this leads to reduction of HIV RNA and DNA levels.Methods:Thirty HIV-infected individuals on ART were randomized to lisinopril at 20mg daily or matching placebo for 24 weeks. All participants underwent rectal biopsies prior to starting the study drug and at 22 weeks, and there were regular blood draws. The primary end point was the change in HIV RNA and DNA levels in rectal tissue. Secondary outcomes included the change in 1) HIV levels in blood; 2) Gag-specific T-cell responses; 3) levels of T-cell activation; and 4) collagen deposition.Results:The addition of lisinopril did not have a significant effect on the levels of HIV RNA or DNA in gut tissue or blood, Gag-specific responses, or levels of T-cell activation. Lisinopril also did not have a significant impact on lymphoid fibrosis in the rectum, as assessed by quantitative histology or heavy water labeling.Conclusions:Treatment with lisinopril for 24 weeks in HIV-infected adults did not have an effect on lymphoid fibrosis, immune activation, or gut tissue viral reservoirs. Further study is needed to see if other anti-fibrotic agents may be useful in reversing lymphoid fibrosis and reducing HIV levels.Clinical Trials Registration:NCT01535235

Memory B Cells, the HLA-B*57 Allele and Natural Control of HIV Infection

Memory B Cells, the HLA-B*57 Allele and Natural Control of HIV Infection