Phenotype, Polyfunctionality, and Antiviral Activity of in vitro Stimulated CD8+ T-Cells From HIV+ Subjects Who Initiated cART at Different Time-Points After Acute Infection.

Since anti-HIV treatment cannot cure the infection, many strategies have been proposed to eradicate the viral reservoir, which still remains as a major challenge. The success of some of these strategies will rely on the ability of HIV-specific CD8+ T-cells (CD8TC) to clear reactivated infected cells. Here, we aimed to investigate the phenotype and function of in vitro expanded CD8TC obtained from HIV+ subjects on combination antiretroviral therapy (cART), either initiated earlier (median = 3 months postinfection, ET: Early treatment) or later (median = 20 months postinfection, DT: Delayed treatment) after infection. Peripheral blood mononuclear cells from 12 DT and 13 ET subjects were obtained and stimulated with Nef and Gag peptide pools plus IL-2 for 14 days. ELISPOT was performed pre- and post-expansion. CD8TC memory/effector phenotype, PD-1 expression, polyfunctionality (CD107a/b, IFN-γ, IL-2, CCL4 (MIP-1ß), and/or TNF-α production) and antiviral activity were evaluated post-expansion. Magnitude of ELISPOT responses increased after expansion by 103 times, in both groups. Expanded cells were highly polyfunctional, regardless of time of cART initiation. The memory/effector phenotype distribution was sharply skewed toward an effector phenotype after expansion in both groups although ET subjects showed significantly higher proportions of stem-cell and central memory CD8TCs. PD-1 expression was clustered in HIV-specific effector memory CD8TCs, subset that also showed the highest proportion of cytokine-producing cells. Moreover, PD-1 expression directly correlated with CD8TC functionality. Expanded CD8TCs from DT and ET subjects were highly capable of mediating antiviral activity, measured by two different assays. Antiviral function directly correlated with the proportion of fully differentiated effector cells (viral inhibition assay) as well as with CD8TC polyfunctionality and PD-1 expression (VITAL assay). In sum, we show that, despite being dampened in subjects on cART, the HIV-specific CD8TC response could be selectively stimulated and expanded in vitro, presenting a high proportion of cells able to carry-out multiple effector functions. Timing of cART initiation had an impact on the memory/effector differentiation phenotype, most likely reflecting how different periods of antigen persistence affected immune function. Overall, these results have important implications for the design and evaluation of strategies aimed at modulating CD8TCs to achieve the HIV functional cure.

Exosomes in HIV infection: A review and critical look.

Exosomes are nanovesicles released into the extracellular medium by different cell types. These vesicles carry a variety of protein and RNA cargos, and have a central role in cellular signaling and regulation. A PubMed search using the term "exosomes" finds 67 articles published in 2006. Ten years later, the same search returns approximately 1200 results for 2016 alone. The growing interest in exosomes within the scientific community reflects the different roles exerted by extracellular vesicles in biological systems and diseases. However, the increase in academic production addressing the biological function of exosomes causes much confusion, especially where the focus is on the role of exosomes in pathological situations. In this review, we critically interpret the current state of the research on exosomes and HIV infection. It is plausible to assume that exosomes influence the pathogenesis of HIV infection through their biological cargo (primarily membrane proteins and microRNAs). On the other hand, evidence for a usurpation of the exosomal budding and trafficking machinery by HIV during infection is limited, although such a mechanism cannot be ruled out. This review also discusses several biological aspects of exosomal function in the immune system. Finally, the limitations of current exosome research are pointed out.

AP-2 Is the Crucial Clathrin Adaptor Protein for CD4 Downmodulation by HIV-1 Nef in Infected Primary CD4+ T Cells.

HIV-1 Nef-mediated CD4 downmodulation involves various host factors. We investigated the importance of AP-1, AP-2, AP-3, V1H-ATPase, ß-COP, and ACOT8 for CD4 downmodulation in HIV-1-infected short hairpin RNA (shRNA)-expressing CD4(+) T cells and characterized direct interaction with Nef by Förster resonance energy transfer (FRET). Binding of lentiviral Nefs to CD4 and AP-2 was conserved, and only AP-2 knockdown impaired Nef-mediated CD4 downmodulation from primary T cells. Altogether, among the factors tested, AP-2 is the most important player for Nef-mediated CD4 downmodulation.

Early Gag immunodominance of the HIV-specific T-cell response during acute/early infection is associated with higher CD8+ T-cell antiviral activity and correlates with preservation of the CD4+ T-cell compartment.

The important role of the CD8(+) T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8(+) T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8(+) T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4(+) T-cell set points were observed in PHI subjects with higher HIV-specific CD8(+) T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1ß (MIP-1ß). All together, this study underscores the importance of CD8(+) T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection.

Higher cross-subtype IFN-γ ELISpot responses to Gag and Nef peptides in Brazilian HIV-1 subtype B- and F1- than in C-infected subjects.

HIV-1 diversity has been considered a huge challenge for the HIV-1 vaccine development. To overcome it, immunogens based on centralized sequences, as consensus, have been tested. In Brazil, the co-circulation of three subtypes offers a suitable scenario to test T cell cross-subtype responses to consensus sequences. Furthermore, we included peptides based on closest viral isolates (CVI) from each subtype analyzed to compare with T cell responses detected against the consensus sequences. The study included 32 subjects infected with HIV-1 subtype B (n=13),C (n=11), and F1 (n=8). Gag and Nef-specific T cell responses were evaluated by IFN-γ-ELISpot assay. Peptides based on CVI sequences were similar to consensus in both reducing genetic distance and detecting T cell responses. A high cross-subtype response between B and F1 in both regions was observed in HIV-1 subtype B and F1-infected subjects. We also found no significant difference in responses to subtype B and C consensus peptides among subtype B-infected subjects. In contrast, the magnitude of T cell responses to consensus C peptides in the Gag region was higher than to consensus B peptides among HIV-1 subtype C-infected subjects. Regarding Nef, subtype C-infected subjects showed higher values to consensus C than to consensus F1 peptides. Moreover, subtype F1-infected subjects presented lower responses to subtype C peptides than to subtype F1 and B. A similar level of responses was detected with group M based peptides in subtype B and F1 infected subjects. However, among subtype C infected subjects, this set of peptides detected lower levels of response than consensus C. Overall, the level of cross-subtype response between subtypes B and F1 was higher than between subtype C and B or C and F1. Our data suggests that the barrier of genetic diversity in HIV-1 group M for vaccine design may be dependent on the subtypes involved.

IL-12 and GM-CSF in DNA/MVA immunizations against HIV-1 CRF12_BF Nef induced T-cell responses with an enhanced magnitude, breadth and quality.

In Argentina, the HIV epidemic is characterized by the co-circulation of subtype B and BF recombinant viral variants. Nef is an HIV protein highly variable among subtypes, making it a good tool to study the impact of HIV variability in the vaccine design setting. We have previously reported a specific cellular response against NefBF with low cross-reactivity to NefB in mice. The aim of this work was to analyze whether the co-administration of IL-12 and GM-CSF, using DNA and MVA vaccine vectors, could improve the final cellular response induced. Mice received three DNA priming doses of a plasmid that express NefBF plus DNAs expressing IL-12 and/or GM-CSF. Afterwards, all the groups were boosted with a MVAnefBF dose. The highest increase in the magnitude of the NefBF response, compared to that induced in the control was found in the IL-12 group. Importantly, a response with higher breadth was detected in groups which received IL-12 or GM-CSF, evidenced as an increased frequency of recognition of homologous (BF) and heterologous (B) Nef peptides, as well as a higher number of other Nef peptide pools representing different viral subtypes. However, these improvements were lost when both DNA cytokines were simultaneously administered, as the response was focused against the immunodominant peptide with a detrimental response towards subdominant epitopes. The pattern of cytokines secreted and the specific-T-cell proliferative capacity were improved in IL-12 and IL-12+GM-CSF groups. Importantly IL-12 generated a significant higher T-cell avidity against a B heterologous peptide.This study indicates that the incorporation of DNA expressing IL-12 in DNA/MVA schemes produced the best results in terms of improvements of T-cell-response key properties such as breadth, cross-reactivity and quality (avidity and pattern of cytokines secreted). These relevant results contribute to the design of strategies aimed to induce T-cell responses against HIV antigens with higher quality.

Current status and perspectives of plant-based candidate vaccines against the human immunodeficiency virus (HIV).

Genetically engineered plants are economical platforms for the large-scale production of recombinant proteins and have been used over the last 21 years as models for oral vaccines against a wide variety of human infectious and autoimmune diseases with promising results. The main inherent advantages of this approach consist in the absence of purification needs and easy production and administration. One relevant infectious agent is the human immunodeficiency virus (HIV), since AIDS evolved as an alarming public health problem implicating very high costs for government agencies in most African and developing countries. The design of an effective and inexpensive vaccine able to limit viral spread and neutralizing the viral entry is urgently needed. Due to the limited efficacy of the vaccines assessed in clinical trials, new HIV vaccines able to generate broad immune profiles are a priority in the field. This review discusses the current advances on the topic of using plants as alternative expression systems to produce functional vaccine components against HIV, including antigens from Env, Gag and early proteins such as Tat and Nef. Ongoing projects of our group based on the expression of chimeric proteins comprising C4 and V3 domains from gp120, as an approach to elicit broadly neutralizing antibodies are mentioned. The perspectives of the revised approaches, such as the great need of assessing the oral immunogenicity and a detailed immunological characterization of the elicited immune responses, are also discussed.

[Development of vaccines for HIV-1. Relevance of subtype-specific cellular immunity]. / Desarrollo de vacunas contra el virus de la inmunodeficiencia humana tipo 1. Relevancia de la inmunidad celular contra subtipos.

It has been almost 30 years since the detection of the first HIV-1 cases and yet an effective and safe vaccine has not been developed. Although, advances in antiretroviral therapy (HAART) have produced a major impact on the pandemic, and even though HIV/aids remains a major concern for developing countries, where access to therapy is limited. The last report from UNAIDS notified 33 million people living with HIV/aids, worldwide, while in Argentina it is estimated that 120,000 persons have been infected. One of the challenges to address and ultimately overcome when developing a vaccine is the high variability of HIV-1. The M group, responsible for the pandemic, is divided into 10 subtypes and several sub-subtypes, in addition to the 48 circulating recombinant forms (CRF) and over one hundred unique recombinant forms (URF). The HIV epidemic in Argentina is as complex as in the rest of the world, characterized by the high prevalence of infections caused by subtype B and BF variants. Despite the wide range of publications focused on the immune response against HIV as well as to vaccine development, how to overcome variability on vaccine antigen selection is still unclear. Studies performed in our laboratory showed the impact of the immunogenicity of BF recombinant variants, both in humans and in animal models. These results are of great concern in vaccine development for our region.

Unexpected diversity of cellular immune responses against Nef and Vif in HIV-1-infected patients who spontaneously control viral replication.

BACKGROUND: HIV-1-infected individuals who spontaneously control viral replication represent an example of successful containment of the AIDS virus. Understanding the anti-viral immune responses in these individuals may help in vaccine design. However, immune responses against HIV-1 are normally analyzed using HIV-1 consensus B 15-mers that overlap by 11 amino acids. Unfortunately, this method may underestimate the real breadth of the cellular immune responses against the autologous sequence of the infecting virus. METHODOLOGY AND PRINCIPAL FINDINGS: Here we compared cellular immune responses against nef and vif-encoded consensus B 15-mer peptides to responses against HLA class I-predicted minimal optimal epitopes from consensus B and autologous sequences in six patients who have controlled HIV-1 replication. Interestingly, our analysis revealed that three of our patients had broader cellular immune responses against HLA class I-predicted minimal optimal epitopes from either autologous viruses or from the HIV-1 consensus B sequence, when compared to responses against the 15-mer HIV-1 type B consensus peptides. CONCLUSION AND SIGNIFICANCE: This suggests that the cellular immune responses against HIV-1 in controller patients may be broader than we had previously anticipated.

Characterization of DNA and MVA vectors expressing Nef from HIV-1 CRF12_BF revealed high immune specificity with low cross-reactivity against subtype B.

The HIV epidemic in Argentina is characterized by the high prevalence of infections caused by subtype B and BF variants. In this study, the Nef protein was used as a tool to study the impact of HIV-1 BF variants in the design of future vaccines. DNA and MVA vectors expressing Nef of the CRF12_BF recombinant form of HIV-1 were generated and characterized. After the administration of single DNAprime/MVAboost immunization schedules in Balb/c mice we found that NefBF delivered from these vectors generated a response of high specificity with low cross-reactivity against subtype B. But, when a more potent response was induced after 3 priming DNA doses and a booster with MVA virus, cross-reactivity against NefB was detected, although of lower magnitude than the NefBF specific. These results will be pivotal for vaccines designs in our region, indicating that antigens from these viral variants must be considered for a future vaccine.

Anti-HIV-1 and anti-HBV immune responses in mice after parenteral and nasal co-administration of a multiantigenic formulation.

The cell-mediated immune response to HIV-1 is an essential element of the mechanisms for viral replication control. Currently, most of the vaccine candidates in clinical trials were developed to stimulate HIV-1-specific CD8+ cytotoxic (CTL) and CD4+ T helper (Th) lymphocytes. We have been working on a novel approach to develop a vaccine formulation for HIV-1 using a recombinant multiepitopic protein (named CR3), which comprises CTL and Th epitope-rich regions of HIV-1 from several subtype B isolates, co-inoculated with the hepatitis B virus surface (HBsAg) and core (HBcAg) antigens of the hepatitis B virus (HBV) as adjuvant. According to our studies in mice, the nasal-subcutaneous co-administration of this multiantigenic formulation induces a strong Th1-biased specific response against CR3, CD8+ T cells in mice spleen and IFN-gamma-secreting cells in mesenteric lymph nodes. Cross-reactive p24-specific IFN-gamma-secreting cells in spleen were also detected. Moreover, Nef-specific antibodies were elicited in mice sera which might avoid the toxic effects of this antigen. However, a marginal anti-CR3 antibody response was elicited in vaginal mucosa. Additionally, we observed anti-HBsAg and anti-HBcAg cellular and humoral responses. In this regard, our multiantigenic formulation might provide immunity against HBV as an additional benefic considering the high HIV-1-HBV co-infection rate reported worldwide.