HIV infection is characterized by a gradual deterioration of immune function mainly in the CD4 compartment. To better understand the dynamics of HIV-specific T cells, we analyzed the kinetics and polyfunctional profiles of Gag-specific CD4+ and CD8+ T cell responses in 12 subtype C-infected individuals with different disease progression profiles from acute to chronic HIV infection. The frequencies of Gag-responsive CD4+ and CD8+ T cells showed distinct temporal kinetics. The peak frequency of Gag-responsive IFNγ+CD4+ T cell was observed at a median of 28 days (IQR: 21-81) post Fiebig I/II staging, whilst Gag-specific IFNγ+CD8+ T cell responses peaked at a median of 253 days (IQR: 136-401 and showed a significant biphasic expansion. The proportion of TNFα-expressing cells within the IFNγ+CD4+ T cell population increased (p=0.001) over time, whilst TNFα-expressing cells within IFNγ+CD8+ T cells declined (p=0.005). Both Gag-responsive CD4+ and CD8+ T cells showed decreased Ki67 expression within the first 120 days post Feibig I/II staging. Prior to the disappearance of Gag-responsive Ki67+CD4+ T cells, these cells positively correlated (p=0.00038) with viremia, indicating that early Gag-responsive CD4 events are shaped by viral burden. No such associations were observed in the Gag-specific CD8+ T cell compartment. Overall, these observations indicate that circulating Gag-responsive CD4+ and CD8+ T cell frequencies and functions are not synchronous and properties change rapidly at different tempos during early HIV infection.
The current study assessed the immunogenicity and protective efficacy of various prime-boost vaccine regimens in rhesus macaques using combinations of recombinant DNA (rDNA), recombinant MVA (rMVA), and subunit gp140 protein. The rDNA and rMVA vectors were constructed to express Env from HIV-1 subtype CRF01_AE and Gag-Pol from CRF01_AE or SIVmac 239. One of the rMVAs, MVA/CMDR, has been recently tested in humans. Immunizations were administered at months 0 and 1 (prime) and months 3 and 6 (boost). After priming, HIV env-specific serum IgG was detected in monkeys receiving gp140 alone or rMVA but not in those receiving rDNA. Titers were enhanced in these groups after boosting either with gp140 alone or with rMVA plus gp140. The groups that received the rDNA prime developed env-specific IgG after boosting with rMVA with or without gp140. HIV Env-specific serum IgG binding antibodies were elicited more frequently and of higher titer, and breadth of neutralizing antibodies was increased with the inclusion of the subunit Env boost. T cell responses were measured by tetramer binding to Gag p11c in Mamu-A*01 macaques, and by IFN-gamma ELISPOT assay to SIV-Gag. T cell responses were induced after vaccination with the highest responses seen in macaques immunized with rDNA and rMVA. Macaques were challenged intravenously with a novel SHIV-E virus (SIVmac239 Gag-Pol with an HIV-1 subtype E-Env CAR402). Post challenge with SHIV-E, antibody titers were boosted in all groups and peaked at 4 weeks. Robust T cell responses were seen in all groups post challenge and in macaques immunized with rDNA and rMVA a clear boosting of responses was seen. A greater than 2 log drop in RNA copies/ml at peak viremia and earlier set point was achieved in macaques primed with rDNA, and boosted with rMVA/SHIV-AE plus gp140. Post challenge viremia in macaques immunized with other regimens was not significantly different to that of controls. These results demonstrate that a gp140 subunit and inclusion of SIV Gag-Pol may be critical for control of SHIV post challenge.
Vaccine regimens using different agents for priming and boosting have become popular for enhancing T cell and Ab responses elicited by candidate HIV/AIDS vaccines. Here we use a simian model to evaluate immunogenicity and protective efficacy of a recombinant modified vaccinia Ankara (MVA) vaccine in the presence and absence of a recombinant DNA prime. The simian vaccines and regimens represent prototypes for candidate HIV vaccines currently undergoing clinical testing.
Recombinant DNA and MVA immunogens expressed simian immunodeficiency virus (SIV)mac239 Gag, PR, RT, and Env sequences. Vaccine schedules tested inoculations of MVA at months 0, 2, and 6 (MMM regimen) or priming with DNA at months 0 and 2 and boosting with MVA at months 4 and 6 (DDMM regimen). Twelve weekly rectal challenges with the heterologous SIV smE660 were initiated at 6 months following the last immunization.
Both regimens elicited similar 61–64% reductions in the per challenge risk of SIVsmE660 transmission despite raising different patterns of immune responses. The DDMM regimen elicited higher magnitudes of CD4 T cells whereas the MMM regimen elicited higher titers and greater avidity Env-specific IgG and more frequent and higher titer SIV-specific IgA in rectal secretions. Both regimens elicited similar magnitudes of CD8 T cells. Magnitudes of T cell responses, specific activities of rectal IgA Ab, and the tested specificities for neutralization and antibody-dependent cellular cytotoxicity did not correlate with risk of infection. However, the avidity of Env-specific IgG had a strong correlation with the per challenge risk of acquisition, but only for the DDMM group.
We conclude that for the tested immunogens in rhesus macaques, the simpler MMM regimen is as protective as the more complex DDMM regimen.
Vaccine; Immunodeficiency virus; Simian immunodeficiency virus; DNA vaccine MVA vaccine; avidity in protection
Characterize responses to a NNRTI-based antiretroviral treatment (ART) initiated during acute HIV infection (AHI).
This was a prospective, single-arm evaluation of once daily, co-formulated emtricitabine/tenofovir/efavirenz initiated during AHI.
The primary endpoint is the proportion of responders with HIV RNA <200 copies/mL by week 24. We examined time-to-viral-suppression and CD8 cell activation in relation to baseline participant characteristics. We compared time-to-viral-suppression and viral dynamics using linear mixed effects models between acutely infected participants and chronically-infected controls.
Between January 2005 and May 2009, 61 AHI participants were enrolled. Of participants whose enrollment date allowed 24 and 48 weeks of follow-up, 47 of 51 (92%) achieved viral suppression to <200 copies/mL by week 24, and 35 of 41 (85.4%) to <50 copies/mL by week 48. The median time from ART initiation to suppression <50 copies/mL was 93 days (range 14–337). Higher HIV RNA levels at ART initiation (p=0.02), but not time from estimated-date-of-infection to ART initiation (p=0.86), were associated with longer time-to-viral-suppression. The median baseline frequency of activated CD8+CD38+HLA-DR+ T-cells was 67% (range 40–95), and was not significantly associated with longer time to viral load suppression (p=0.15). Viremia declined to <50 copies/mL more rapidly in AHI than chronically-infected participants. Mixed model analysis demonstrated similar phase I HIV RNA decay rates between acute and chronically-infected participants, and more rapid viral decline in acutely-infected participants in phase II.
Once daily emtricitabine/tenofovir/efavirenz initiated during AHI achieves rapid and sustained HIV suppression during this highly infectious period.
Acute HIV infection; NNRTIs; antiretroviral therapy; immune activation; viral dynamics
Most antibodies that broadly neutralize HIV-1 are highly somatically mutated in antibody clonal lineages that persist over time. Here, we describe the analysis of human antibodies induced during an HIV-1 vaccine trial (GSK PRO HIV-002) that used the clade B envelope (Env) gp120 of clone W6.1D (gp120W6.1D). Using dual-color antigen-specific sorting, we isolated Env-specific human monoclonal antibodies (MAbs) and studied the clonal persistence of antibodies in the setting of HIV-1 Env vaccination. We found evidence of VH somatic mutation induced by the vaccine but only to a modest level (3.8% ± 0.5%; range 0 to 8.2%). Analysis of 34 HIV-1-reactive MAbs recovered over four immunizations revealed evidence of both sequential recruitment of naïve B cells and restimulation of previously recruited memory B cells. These recombinant antibodies recapitulated the anti-HIV-1 activity of participant serum including pseudovirus neutralization and antibody-dependent cell-mediated cytotoxicity (ADCC). One antibody (3491) demonstrated a change in specificity following somatic mutation with binding of the inferred unmutated ancestor to a linear C2 peptide while the mutated antibody reacted only with a conformational epitope in gp120 Env. Thus, gp120W6.1D was strongly immunogenic but over four immunizations induced levels of affinity maturation below that of broadly neutralizing MAbs. Improved vaccination strategies will be needed to drive persistent stimulation of antibody clonal lineages to induce affinity maturation that results in highly mutated HIV-1 Env-reactive antibodies.
HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8+ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell–mediated in vivo control of HIV-1. Primary HIV-1–specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or “vertical” immunodominance as the primary determinant of in vivo CD8+ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8+ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance.
CD8-mediated virus inhibition can be detected in HIV-1-positive subjects who naturally control virus replication. Characterizing the inhibitory function of CD8+ T cells during acute HIV-1 infection (AHI) can elucidate the nature of the CD8+ responses that can be rapidly elicited and that contribute to virus control. We examined the timing and HIV-1 antigen specificity of antiviral CD8+ T cells during AHI. Autologous and heterologous CD8+ T cell antiviral functions were assessed longitudinally during AHI in five donors from the CHAVI 001 cohort using a CD8+ T cell-mediated virus inhibition assay (CD8 VIA) and transmitted/founder (T/F) viruses. Potent CD8+ antiviral responses against heterologous T/F viruses appeared during AHI at the first time point sampled in each of the 5 donors (Fiebig stages 1/2 to 5). Inhibition of an autologous T/F virus was durable to 48 weeks; however, inhibition of heterologous responses declined concurrent with the resolution of viremia. HIV-1 viruses from 6 months postinfection were more resistant to CD8+-mediated virus inhibition than cognate T/F viruses, demonstrating that the virus escapes early from CD8+ T cell-mediated inhibition of virus replication. CD8+ T cell antigen-specific subsets mediated inhibition of T/F virus replication via soluble components, and these soluble responses were stimulated by peptide pools that include epitopes that were shown to drive HIV-1 escape during AHI. These data provide insights into the mechanisms of CD8-mediated virus inhibition and suggest that functional analyses will be important for determining whether similar antigen-specific virus inhibition can be induced by T cell-directed vaccine strategies.
A modest change in HIV-1 fitness can have a significant impact on viral quasispecies evolution and viral pathogenesis, transmission and disease progression. To determine the impact of immune escape mutations selected by cytotoxic T lymphocytes (CTL) on viral fitness in the context of the cognate transmitted/founder (T/F) genome, we developed a new competitive fitness assay using molecular clones of T/F genomes lacking exogenous genetic markers and a highly sensitive and precise parallel allele-specific sequencing (PASS) method.
The T/F and mutant viruses were competed in CD4+ T-cell enriched cultures, relative proportions of viruses were assayed after repeated cell-free passage, and fitness costs were estimated by mathematical modeling. Naturally occurring HLA B57-restricted mutations involving the TW10 epitope in Gag and two epitopes in Tat/Rev and Env were assessed independently and together. Compensatory mutations which restored viral replication fitness were also assessed. A principal TW10 escape mutation, T242N, led to a 42% reduction in replication fitness but V247I and G248A mutations in the same epitope restored fitness to wild-type levels. No fitness difference was observed between the T/F and a naturally selected variant carrying the early CTL escape mutation (R355K) in Env and a reversion mutation in the Tat/Rev overlapping region.
These findings reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations, and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. Analysis of T/F genomes and their evolved progeny is a powerful approach for assessing the impact of composite mutational events on viral fitness.
Human immunodeficiency virus type I; Viral fitness; Cytotoxic T lymphocytes; Immune escape mutation; Transmitted/founder virus; Mathematical model
In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case–control analysis to identify antibody and cellular immune correlates of infection risk.
In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up.
Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P = 0.02; q = 0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P = 0.03; q = 0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies.
This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.
Preclinical studies of HIV-1 vaccine candidates have typically shown post-infection virologic control, but protection against acquisition of infection has previously only been reported against neutralization-sensitive virus challenges1–3. Here we demonstrate vaccine protection against acquisition of fully heterologous, neutralization-resistant virus challenges in rhesus monkeys. Adenovirus/poxvirus and adenovirus/adenovirus vector-based vaccines expressing SIVsmE543 Gag, Pol, and Env antigens resulted in a ≥80% reduction in the per-exposure probability of infection4,5 against repetitive, intrarectal SIVmac251 challenges in rhesus monkeys. Protection against acquisition of infection exhibited distinct immunologic correlates as compared with post-infection virologic control and required the inclusion of Env in the vaccine regimen. These data demonstrate the first proof-of-concept that optimized HIV-1 vaccine candidates can block acquisition of stringent, heterologous, neutralization-resistant virus challenges in rhesus monkeys.
A simian immunodeficiency virus (SIV) vaccine coexpressing granulocyte-macrophage colony stimulating factor (GM-CSF) prevented infection in 71% of macaques that received 12 rectal challenges. The SIVsmE660 challenge had the tropism of incident human immunodeficiency virus (HIV) infections and a similar genetic distance from the SIV239 vaccine as intraclade HIV isolates. The heterologous prime-boost vaccine regimen used recombinant DNA for priming and recombinant modified vaccinia Ankara for boosting. Co-expression of GM-CSF in the DNA prime enhanced the avidity of elicited immunoglobulin G for SIV envelope glycoproteins, the titers of neutralizing antibody for easy-to-neutralize SIV isolates, and antibody-dependent cellular cytotoxicity. Impressively, the co-expressed GM-CSF increased vaccine-induced prevention of infection from 25% in the non–GM-CSF co-expressing vaccine group to 71% in the GM-CSF co-expressing vaccine group. The prevention of infection showed a strong correlation with the avidity of the elicited Env-specific antibody for the Env of the SIVsmE660 challenge virus (r = 0.9; P < .0001).
Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional antibody responses are present in breast milk, and these mucosal antibody responses may play a role in protection of the majority of HIV-exposed, breastfeeding infants. Therefore, characterization of HIV-specific antibodies produced by B cells in milk could guide the development of vaccines that elicit protective mucosal antibody responses.
We isolated B cells from colostrum of an HIV-infected lactating woman with a detectable neutralization response in milk and recombinantly produced and characterized the resulting HIV-1 Envelope (Env)-specific monoclonal antibodies (mAbs).
The identified HIV-1 Env-specific colostrum mAbs, CH07 and CH08, represent two of the first mucosally-derived anti-HIV antibodies yet to be reported. Colostrum mAb CH07 is a highly-autoreactive, weakly-neutralizing gp140-specific mAb that binds to linear epitopes in the gp120 C5 region and gp41 fusion domain. In contrast, colostrum mAb CH08 is a nonpolyreactive CD4-inducible (CD4i) gp120-specific mAb with moderate breadth of neutralization.
These novel HIV-neutralizing mAbs isolated from a mucosal compartment provide insight into the ability of mucosal B cell populations to produce functional anti-HIV antibodies that may contribute to protection against virus acquisition at mucosal surfaces.
We sought to identify biomarker responses to tuberculosis specific antigens which could 1) improve the diagnosis of tuberculosis infection and 2) allow the differentiation of active and latent infections. Seventy subjects with active tuberculosis (N=12), latent tuberculosis (N=32), or no evidence of tuberculosis infection (N=26) were evaluated. We used the Luminex Multiplexed Bead Array platform to simultaneously evaluate 25 biomarkers in the supernatant of whole blood samples following overnight stimulation using the Quantiferon® Gold In-Tube kit. We defined the response to stimulation as the difference (within an individual patient) between the response to the pooled tuberculosis antigens and the negative control. IP-10 response was significantly higher in tuberculosis-infected (active or latent) subjects compared to the uninfected group (p <0.0001). Among the 25 parameters, expression levels of IL-15 and MCP-1 were found to be significantly higher in the active tuberculosis group compared to the latent tuberculosis group (p = 0.0006 and 0.0030, respectively). When combined, IL-15 and MCP-1 accurately identified 83% of active and 88% of latent infections. The combination of IL-15 and MCP-1 responses was accurate in distinguishing persons with active tuberculosis from persons with latent tuberculosis in this study.
tuberculosis; immune response; diagnosis; biomarker
HIV-1 often evades cytotoxic T cell (CTL) responses by generating variants that are not recognized by CTLs. We used single-genome amplification and sequencing of complete HIV genomes to identify longitudinal changes in the transmitted/founder virus from the establishment of infection to the viral set point at 1 year after the infection. We found that the rate of viral escape from CTL responses in a given patient decreases dramatically from acute infection to the viral set point. Using a novel mathematical model that tracks the dynamics of viral escape at multiple epitopes, we show that a number of factors could potentially contribute to a slower escape in the chronic phase of infection, such as a decreased magnitude of epitope-specific CTL responses, an increased fitness cost of escape mutations, or an increased diversity of the CTL response. In the model, an increase in the number of epitope-specific CTL responses can reduce the rate of viral escape from a given epitope-specific CTL response, particularly if CD8+ T cells compete for killing of infected cells or control virus replication nonlytically. Our mathematical framework of viral escape from multiple CTL responses can be used to predict the breadth and magnitude of HIV-specific CTL responses that need to be induced by vaccination to reduce (or even prevent) viral escape following HIV infection.
Despite months of mucosal virus exposure, the majority of breastfed infants born to HIV-infected mothers do not become infected, raising the possibility that immune factors in milk inhibit mucosal transmission of HIV. HIV Envelope (Env)-specific antibodies are present in the milk of HIV-infected mothers, but little is known about their virus-specific functions. In this study, HIV Env-specific antibody binding, autologous and heterologous virus neutralization, and antibody-dependent cell cytotoxicity (ADCC) responses were measured in the milk and plasma of 41 HIV-infected lactating women. Although IgA is the predominant antibody isotype in milk, HIV Env-specific IgG responses were higher in magnitude than HIV Env-specific IgA responses in milk. The concentrations of anti-HIV gp120 IgG in milk and plasma were directly correlated (r = 0.75; P < 0.0001), yet the response in milk was 2 logarithm units lower than in plasma. Similarly, heterologous virus neutralization (r = 0.39; P = 0.010) and ADCC activity (r = 0.64; P < 0.0001) in milk were directly correlated with that in the systemic compartment but were 2 log units lower in magnitude. Autologous neutralization was rarely detected in milk. Milk heterologous virus neutralization titers correlated with HIV gp120 Env-binding IgG responses but not with IgA responses (r = 0.71 and P < 0.0001, and r = 0.17 and P = 0.30). Moreover, IgGs purified from milk and plasma had equal neutralizing potencies against a tier 1 virus (r = 0.65; P < 0.0001), whereas only 1 out of 35 tested non-IgG milk fractions had detectable neutralization. These results suggest that plasma-derived IgG antibodies mediate the majority of the low-level HIV neutralization and ADCC activity in breast milk.
Functional immunologic assays using cryopreserved peripheral blood mononuclear cells (PBMC) are influenced by blood processing, storage and shipment. The objective of this study was to compare the viability, recovery and ELISPOT results of PBMC stored and shipped in liquid nitrogen (LN/LN) or stored in LN and shipped on dry ice (LN/DI) or stored at −70°C for 3 to 12 weeks and shipped on DI (70/DI 3 to 12); and to assess the effect of donor HIV infection status on the interaction between storage/shipment and the outcome measures. PBMC from 12 HIV-infected and 12 uninfected donors showed that LN/LN conferred higher viability and recovery than LN/DI or 70/DI 3, 6, 9 or 12. LN/DI PBMC had higher viability than any 70/DI PBMC. The PBMC viability and recovery linearly decreased with the duration of storage at −70°C from 3 to 12 weeks. This effect was more pronounced in samples from HIV-infected than uninfected donors. Results of ELISPOT assays using CMV pp65, CEF and Candida albicans antigens were qualitatively and quantitatively similar across LN/LN, LN/DI and 70/DI 3. However, ELISPOT values significantly decreased with the duration of storage at −70°C both in HIV-infected and uninfected donors. ELISPOT results also decreased with PBMC viability <70%.
Purpose of review
New findings continue to support the notion that broadly crossreactive neutralizing antibody induction is a worthwhile and achievable goal for HIV-1 vaccines. Immunogens are needed that can overcome the genetic variability and complex immune evasion tactics of the virus. Other antibodies might bridge innate and acquired immunity for possible beneficial vaccine effects. This review summarizes progress made over the past year that has enhanced our understanding of humoral immunity as it relates to HIV-1 vaccine development.
Although a clear path to designing an effective neutralizing antibody-based HIV-1 vaccine remains elusive, there is new information on how antibodies neutralize HIV-1, the epitopes involved, and clues to the possible nature of protective immunogens that keep this goal alive. Moreover, there is a greater understanding of HIV-1 diversity and its possible limits under immune pressure. Other antibodies might possess antiviral activity by mechanisms involving Fc receptor engagement or complement activation that would be of value for HIV-1 vaccines.
Recent developments strengthen the rationale for antibody-based HIV-1 vaccine immunogens and provide a stronger foundation for vaccine discovery.
adjuvants; AIDS; antibodies; complement; Fc receptors; vaccines
Among nonneutralizing HIV-1 envelope antibodies (Abs), those capable of mediating antibody-dependent cellular cytotoxicity (ADCC) activity have been postulated to be important for control of HIV-1 infection. ADCC-mediating Ab must recognize HIV-1 antigens expressed on the membrane of infected cells and bind the Fcγ receptor (FcR) of the effector cell population. However, the precise targets of serum ADCC antibody are poorly characterized. The human monoclonal antibody (MAb) A32 is a nonneutralizing antibody isolated from an HIV-1 chronically infected person. We investigated the ability of MAb A32 to recognize HIV-1 envelope expressed on the surface of CD4+ T cells infected with primary and laboratory-adapted strains of HIV-1, as well as its ability to mediate ADCC activity. The MAb A32 epitope was expressed on the surface of HIV-1-infected CD4+ T cells earlier than the CD4-inducible (CD4i) epitope bound by MAb 17b and the gp120 carbohydrate epitope bound by MAb 2G12. Importantly, MAb A32 was a potent mediator of ADCC activity. Finally, an A32 Fab fragment blocked the majority of ADCC-mediating Ab activity in plasma of subjects chronically infected with HIV-1. These data demonstrate that the epitope defined by MAb A32 is a major target on gp120 for plasma ADCC activity.
Acute HIV infection (AHI) is a critical phase of infection when irreparable damage to the immune system occurs and subjects are very infectious. We studied subjects with AHI prospectively to develop better treatment and public health interventions.
Cross-sectional screening was employed to detect HIV RNA positive, antibody negative subjects. Date of HIV acquisition was estimated from clinical history and correlated with sequence diversity assessed by single genome amplification (SGA). Twenty-two cytokines/chemokines were measured from enrollment through week 24.
Thirty-seven AHI subjects were studied. In 7 participants with limited exposure windows, the median exposure to HIV occurred 14 days before symptom onset. Lack of viral sequence diversification confirmed the short duration of infection. Transmission dates estimated by SGA/sequencing using molecular clock models correlated with transmission dates estimated by symptom onset in individuals infected with single HIV variants (mean of 28 versus 33 days). Only 10 of 22 cytokines/chemokines were significantly elevated among AHI participants at enrollment compared to uninfected controls, and only 4 participants remained seronegative at enrollment.
The results emphasize the difficulty in recruiting subjects early in AHI. Viral sequence diversity proved accurate in estimating time of infection. Regardless of aggressive screening, peak viremia and inflammation occurred before enrollment and potential intervention. Given the personal and public health importance, improved AHI detection is urgently needed.
The hallmark of chronic viral infections is a progressive exhaustion of antigen specific CD8+ T cells that leads to persisting viral replication. It is generally believed that exhaustion is a consequence of the accumulation of multiple inhibitory receptors on CD8+ T cells that makes them dysfunctional. Here we show that during human chronic HIV-1 infection a CD8+ T cell positive costimulatory pathway mediated by DNAM-1 is also disrupted. Thus, DNAM-1 downregulation on CD8+ T cells aggravates the impairment of CTL effector function in chronic HIV-1 infection.
HIV-1; exhaustion; co-stimulation
We evaluated replication-defective poxvirus vectors (modified vaccinia Ankara [MVA] and fowlpox [FPV]) in a homologous and heterologous vector prime-boost vaccination regimen containing matching HIV inserts (MVA-HIV and FPV-HIV) given at months 0, 1, 3, 5 and 7 in 150 healthy HIV-negative vaccinia-naïve participants. FPV-HIV alone was poorly immunogenic, while the high dose (109 pfu/2ml) of MVA-HIV alone elicited maximal responses after two injections: CD4+ and CD8+ T-cell responses in 26/55 (47.3%) and 5/60 (8.3%) of participants, respectively and IFN-γ ELISpot responses in 28/62 (45.2%). The infrequent CD8+ T-cell responses following MVA-HIV priming were boosted only by the heterologous (FPV-HIV) construct in 14/27 [51.9%] of participants post-4th vaccination. Alternatively, HIV envelope-specific binding antibodies were demonstrated in approximately two-thirds of recipients of the homologous boosting regimen, but in less than 20% of subjects after the heterologous vector boost. Thus, a heterologous poxvirus vector prime-boost regimen can induce an HIV-specific CD8+ T-cell and CD4+ T-cell responses, which may be an important feature of an optimal regimen for preventive HIV vaccination.
Preventive HIV vaccine; MVA and Fowlpox vectors; Phase I clinical trial
In the present study, we analyzed the functional profile of CD8+ T-cell responses directed against autologous transmitted/founder HIV-1 isolates during acute and early infection, and examined whether multifunctionality is required for selection of virus escape mutations. Seven anti-retroviral therapy-naïve subjects were studied in detail between 1 and 87 weeks following onset of symptoms of acute HIV-1 infection. Synthetic peptides representing the autologous transmitted/founder HIV-1 sequences were used in multiparameter flow cytometry assays to determine the functionality of HIV-1-specific CD8+ T memory cells. In all seven patients, the earliest T cell responses were predominantly oligofunctional, although the relative contribution of multifunctional cell responses increased significantly with time from infection. Interestingly, only the magnitude of the total and not of the poly-functional T-cell responses was significantly associated with the selection of escape mutants. However, the high contribution of MIP-1β-producing CD8+ T-cells to the total response suggests that mechanisms not limited to cytotoxicity could be exerting immune pressure during acute infection. Lastly, we show that epitope entropy, reflecting the capacity of the epitope to tolerate mutational change and defined as the diversity of epitope sequences at the population level, was also correlated with rate of emergence of escape mutants.
An important role for the polyfunctional T-cell fraction of anti-HIV CD8 responses during chronic HIV infection has previously been suggested. This study characterized the role of polyfunctional T-cells directed against the transmitted/founder virus in the selection of viral escape mutants during acute HIV-1 infection within a unique cohort of individuals recruited within 3 weeks from the onset of symptoms at the time when the virus load was still declining. For the first time, the sequences of the transmitted/founder virus isolated from each patient were used. Interestingly, polyfunctionality was not found to be a pre-requisite for selection of escape mutations. A novel significant correlation is found between the order of appearance of escape mutations in different epitope sequences and both the magnitude of the CD8+ T-cell responses and the degree of entropy of the individual epitopes. A high proportion of the T-cells participating in the total response produced MIP-1β, suggesting that mechanisms not limited to the killing of infected cells might play a relevant role in early infection. This highlights the importance of measuring the quality of the CD8+ lymphocyte response and the sequence of the transmitted virus isolates to better understand the mechanisms of control of HIV replication during acute infection.
Control of HIV-1 replication following nonsterilizing HIV-1 vaccination could be achieved by vaccine-elicited CD8+ T-cell-mediated antiviral activity. To date, neither the functional nor the phenotypic profiles of CD8+ T cells capable of this activity are clearly understood; consequently, little is known regarding the ability of vaccine strategies to elicit them. We used multiparameter flow cytometry and viable cell sorts from phenotypically defined CD8+ T-cell subsets in combination with a highly standardized virus inhibition assay to evaluate CD8+ T-cell-mediated inhibition of viral replication. Here we show that vaccination against HIV-1 Env and Gag-Pol by DNA priming followed by recombinant adenovirus type 5 (rAd5) boosting elicited CD8+ T-cell-mediated antiviral activity against several viruses with either lab-adapted or transmitted virus envelopes. As it did for chronically infected virus controllers, this activity correlated with HIV-1-specific CD107a or macrophage inflammatory protein 1β (MIP-1β) expression from HIV-1-specific T cells. Moreover, for vaccinees or virus controllers, purified memory CD8+ T cells from a wide range of differentiation stages were capable of significantly inhibiting virus replication. Our data define attributes of an antiviral CD8+ T-cell response that may be optimized in the search for an efficacious HIV-1 vaccine.
The prevailing paradigm of T lymphocyte control of viral replication is that the protective capacity of virus-specific CD8+ T cells is directly proportional to the number of functions they can perform, with IL-2 production capacity considered critical. Having recently defined rapid perforin upregulation as a novel effector function of antigen-specific CD8+ T cells, here we sought to determine whether new perforin production is a component of polyfunctional CD8+ T cell responses that contributes to the control of several human viral infections: cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza (flu), and adenovirus (Ad). We stimulated normal human donor PBMC with synthetic peptides whose amino acid sequences correspond to defined CTL epitopes in the aforementioned viruses, and then used polychromatic flow cytometry to measure the functional capacity and the phenotype of the responding CD8+ T cells. While EBV and flu-specific CD8+ T cells rarely upregulate perforin, CMV-specific cells often do and Ad stimulates an exceptionally strong perforin response. The differential propensity of CD8+ T cells to produce either IL-2 or perforin is in part related to levels of CD28 and the transcription factor T-bet, as CD8+ T cells that rapidly upregulate perforin harbor high levels of T-bet and those producing IL-2 express high amounts of CD28. Thus, “polyfunctional” profiling of antigen-specific CD8+ T cells must not be limited to simply the number of functions the cell can perform, or one particular memory phenotype, but should actually define which combinations of memory markers and functions are relevant in each pathogenic context.
Although CD8+ T cells are thought to be largely responsible for the control of viral infections, exactly how they mediate protection is uncertain. One approach to assessing their protective capacity is to measure several of their functions simultaneously. Generally, it is believed the more functions a cell can perform, the better its potential to control viral replication. A multi-functional response including interleukin-2 (IL-2) production is currently valued as the key correlate of protection. We recently characterized a novel CD8+ T cell function: rapid perforin upregulation, which serves to contribute to and sustain the killing of virally infected host cells. In this study, we show that new perforin is abundant during adenovirus and cytomegalovirus infections, but scarcely detected in the context of influenza and Epstein-Barr virus. Importantly, perforin and IL-2 are rarely co-expressed. The significance of this relationship is that we can no longer assume the more functions a CD8+ T cell performs in response to a virus the better. Thus, when considering vaccine design, no single functional profile will likely be protective across all pathogens. Rather, vaccine-induced T cell responses may need to be “pathogen-specific”, as different T cell functional responses will be important for controlling different viral infections.
There is an urgent need for a vaccine capable of preventing HIV infection or the development of HIV-related disease. A number of approaches designed to stimulate HIV-specific CD8+ cytotoxic T cell responses together with helper responses are presently under evaluation. In this phase 1, multi-center, placebo-controlled trial, we tested the ability of a novel multi-epitope peptide vaccine to elicit HIV-specific immunity. To enhance the immunogenicity of the peptide vaccine, half of the vaccine recipients received recombinant GM-CSF protein as a co-adjuvant. The vaccine was safe; tolerability was moderate, with a number of adverse events related to local injection site reactogenicity. Anti-GM-CSF antibody responses developed in the majority of GM-CSF recipients but were not associated with adverse hematologic events. The vaccine was only minimally immunogenic. Six of 80 volunteers who received vaccine developed HIV-specific responses as measured by interferon-gamma ELISPOT assay, and measurable responses were transient. This study failed to demonstrate that GM-CSF can substantially improve the overall weak immunogenicity of a multiepitope peptide-based HIV vaccine.