ABSTRACT
UNLABELLED: In a follow-up to the modest efficacy observed in the RV144 trial, researchers in the HIV vaccine field seek to substantiate and extend the results by evaluating other poxvirus vectors and combinations with DNA and protein vaccines. Earlier clinical trials (EuroVacc trials 01 to 03) evaluated the immunogenicity of HIV-1 clade C GagPolNef and gp120 antigens delivered via the poxviral vector NYVAC. These showed that a vaccination regimen including DNA-C priming prior to a NYVAC-C boost considerably enhanced vaccine-elicited immune responses compared to those with NYVAC-C alone. Moreover, responses were improved by using three as opposed to two DNA-C primes. In the present study, we assessed in nonhuman primates whether such vaccination regimens can be streamlined further by using fewer and accelerated immunizations and employing a novel generation of improved DNA-C and NYVAC-C vaccine candidates designed for higher expression levels and more balanced immune responses. Three different DNA-C prime/NYVAC-C+ protein boost vaccination regimens were tested in rhesus macaques. All regimens elicited vigorous and well-balanced CD8(+)and CD4(+)T cell responses that were broad and polyfunctional. Very high IgG binding titers, substantial antibody-dependent cellular cytotoxicity (ADCC), and modest antibody-dependent cell-mediated virus inhibition (ADCVI), but very low neutralization activity, were measured after the final immunizations. Overall, immune responses elicited in all three groups were very similar and of greater magnitude, breadth, and quality than those of earlier EuroVacc vaccines. In conclusion, these findings indicate that vaccination schemes can be simplified by using improved antigens and regimens. This may offer a more practical and affordable means to elicit potentially protective immune responses upon vaccination, especially in resource-constrained settings. IMPORTANCE: Within the EuroVacc clinical trials, we previously assessed the immunogenicity of HIV clade C antigens delivered in a DNA prime/NYVAC boost regimen. The trials showed that the DNA prime crucially improved the responses, and three DNA primes with a NYVAC boost appeared to be optimal. Nevertheless, T cell responses were primarily directed toward Env, and humoral responses were modest. The aim of this study was to assess improved antigens for the capacity to elicit more potent and balanced responses in rhesus macaques, even with various simpler immunization regimens. Our results showed that the novel antigens in fact elicited larger numbers of T cells with a polyfunctional profile and a good Env-GagPolNef balance, as well as high-titer and Fc-functional antibody responses. Finally, comparison of the different schedules indicates that a simpler regimen of only two DNA primes and one NYVAC boost in combination with protein may be very efficient, thus showing that the novel antigens allow for easier immunization protocols.
Subject(s)
AIDS Vaccines/immunology , DNA Primers , HIV-1/immunology , Vaccines, DNA/immunology , AIDS Vaccines/genetics , Animals , Antibodies, Neutralizing/immunology , Antibody-Dependent Cell Cytotoxicity , HIV Antibodies/immunology , HIV Antigens/immunology , Interferon-gamma/biosynthesis , Male , T-Lymphocytes/immunology , Vaccination/methods , Vaccines, DNA/genetics , gag Gene Products, Human Immunodeficiency Virus/genetics , gag Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
UNLABELLED: We compared the HIV-1-specific cellular and humoral immune responses elicited in rhesus macaques immunized with two poxvirus vectors (NYVAC and ALVAC) expressing the same HIV-1 antigens from clade C, Env gp140 as a trimeric cell-released protein and a Gag-Pol-Nef polyprotein as Gag-induced virus-like particles (VLPs) (referred to as NYVAC-C and ALVAC-C). The immunization protocol consisted of two doses of the corresponding poxvirus vector plus two doses of a combination of the poxvirus vector and a purified HIV-1 gp120 protein from clade C. This immunogenicity profile was also compared to that elicited by vaccine regimens consisting of two doses of the ALVAC vector expressing HIV-1 antigens from clades B/E (ALVAC-vCP1521) plus two doses of a combination of ALVAC-vCP1521 and HIV-1 gp120 protein from clades B/E (similar to the RV144 trial regimen) or clade C. The results showed that immunization of macaques with NYVAC-C stimulated at different times more potent HIV-1-specific CD4(+) T-cell responses and induced a trend toward higher-magnitude HIV-1-specific CD8(+) T-cell immune responses than did ALVAC-C. Furthermore, NYVAC-C induced a trend toward higher levels of binding IgG antibodies against clade C HIV-1 gp140, gp120, or murine leukemia virus (MuLV) gp70-scaffolded V1/V2 and toward best cross-clade-binding IgG responses against HIV-1 gp140 from clades A, B, and group M consensus, than did ALVAC-C. Of the linear binding IgG responses, most were directed against the V3 loop in all immunization groups. Additionally, NYVAC-C and ALVAC-C also induced similar levels of HIV-1-neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC) responses. Interestingly, binding IgA antibody levels against HIV-1 gp120 or MuLV gp70-scaffolded V1/V2 were absent or very low in all immunization groups. Overall, these results provide a comprehensive survey of the immunogenicity of NYVAC versus ALVAC expressing HIV-1 antigens in nonhuman primates and indicate that NYVAC may represent an alternative candidate to ALVAC in the development of a future HIV-1 vaccine. IMPORTANCE: The finding of a safe and effective HIV/AIDS vaccine immunogen is one of the main research priorities. Here, we generated two poxvirus-based HIV vaccine candidates (NYVAC and ALVAC vectors) expressing the same clade C HIV-1 antigens in separate vectors, and we analyzed in nonhuman primates their immunogenicity profiles. The results showed that immunization with NYVAC-C induced a trend toward higher HIV-1-specific cellular and humoral immune responses than did ALVAC-C, indicating that this new NYVAC vector could be a novel optimized HIV/AIDS vaccine candidate for human clinical trials.
Subject(s)
AIDS Vaccines/immunology , Gene Products, env/metabolism , Genetic Vectors/immunology , HIV Infections/prevention & control , Vaccines, Synthetic/immunology , Viral Vaccines/immunology , Animals , Antibodies, Neutralizing , Chick Embryo , HIV Antibodies , HIV Antigens/metabolism , Macaca mulatta , Poxviridae/genetics , Promoter Regions, Genetic/genetics , Viral Plaque AssayABSTRACT
A successful HIV vaccine will likely induce both humoral and cell-mediated immunity, however, the enormous diversity of HIV has hampered the development of a vaccine that effectively elicits both arms of the adaptive immune response. To tackle the problem of viral diversity, T cell-based vaccine approaches have focused on two main strategies (i) increasing the breadth of vaccine-induced responses or (ii) increasing vaccine-induced responses targeting only conserved regions of the virus. The relative extent to which set-point viremia is impacted by epitope-conservation of CD8(+) T cell responses elicited during early HIV-infection is unknown but has important implications for vaccine design. To address this question, we comprehensively mapped HIV-1 CD8(+) T cell epitope-specificities in 23 ART-naïve individuals during early infection and computed their conservation score (CS) by three different methods (prevalence, entropy and conseq) on clade-B and group-M sequence alignments. The majority of CD8(+) T cell responses were directed against variable epitopes (p<0.01). Interestingly, increasing breadth of CD8(+) T cell responses specifically recognizing conserved epitopes was associated with lower set-point viremia (râ=â- 0.65, pâ=â0.009). Moreover, subjects possessing CD8(+) T cells recognizing at least one conserved epitope had 1.4 log10 lower set-point viremia compared to those recognizing only variable epitopes (pâ=â0.021). The association between viral control and the breadth of conserved CD8(+) T cell responses may be influenced by the method of CS definition and sequences used to determine conservation levels. Strikingly, targeting variable versus conserved epitopes was independent of HLA type (pâ=â0.215). The associations with viral control were independent of functional avidity of CD8(+) T cell responses elicited during early infection. Taken together, these data suggest that the next-generation of T-cell based HIV-1 vaccines should focus on strategies that can elicit CD8(+) T cell responses to multiple conserved epitopes of HIV-1.
Subject(s)
Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , Conserved Sequence , Epitopes, T-Lymphocyte/immunology , HIV Infections/immunology , HIV-1/immunology , Immunity, Cellular , Amino Acid Sequence , Antigens, Viral/genetics , CD8-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/virology , Epitope Mapping , Epitopes, T-Lymphocyte/genetics , Gene Expression , Genetic Heterogeneity , HIV Infections/pathology , HIV Infections/virology , HIV-1/growth & development , Homosexuality, Male , Humans , Male , Molecular Sequence Data , Viral Load/immunology , Virus Replication/immunologyABSTRACT
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.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes, T-Lymphocyte/immunology , HIV Infections/immunology , HIV-1/immunology , Immune Evasion , Immunity, Cellular , Adolescent , Adult , CD8-Positive T-Lymphocytes/pathology , Female , HIV Infections/pathology , Humans , MaleABSTRACT
Vaccination and SIV challenge of macaque species is the best animal model for evaluating candidate HIV vaccines in pre-clinical studies. As such, robust assays optimized for use in nonhuman primates are necessary for reliable ex vivo measurement of immune responses and identification of potential immune correlates of protection. We optimized and qualified an 8-color intracellular cytokine staining assay for the measurement of IFNγ, IL-2, and TNF from viable CD4 and CD8 T cells from cryopreserved rhesus macaque PBMC stimulated with peptides. After optimization, five laboratories tested assay performance using the same reagents and PBMC samples; similar results were obtained despite the use of flow cytometers with different configurations. The 8-color assay was then subjected to a pre-qualification study to quantify specificity and precision. These data were used to set positivity thresholds and to design the qualification protocol. Upon completion of the qualification study, the assay was shown to be highly reproducible with low inter-aliquot, inter-day, and inter-operator variability according to the qualification criteria with an overall variability of 20-40% for each outcome measurement. Thus, the 8-color ICS assay was formally qualified according to the ICH guidelines Q2 (R1) for specificity and precision indicating that it is considered a standardized/robust assay acceptable for use in pre-clinical trial immunogenicity testing.
Subject(s)
AIDS Vaccines/administration & dosage , Cytokines/analysis , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , Staining and Labeling/methods , T-Lymphocytes/immunology , Animals , Cell Separation , Color , Disease Models, Animal , Drug Evaluation, Preclinical , Flow Cytometry/methods , Guidelines as Topic , Humans , Intracellular Space , Macaca mulatta , Observer Variation , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
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-1ß-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.
Subject(s)
Antigenic Variation/immunology , CD8-Positive T-Lymphocytes/immunology , Epitopes/immunology , HIV Infections/immunology , HIV-1/genetics , HIV-1/immunology , Immune Evasion/genetics , Adult , Antigenic Variation/genetics , CD8-Positive T-Lymphocytes/metabolism , Epitopes/genetics , HIV Infections/virology , Humans , Immune Evasion/immunology , Immunologic Memory/immunology , Immunologic Memory/physiology , Male , Middle Aged , Mutation/physiology , Organisms, Genetically Modified , Selection, Genetic/immunology , Young AdultABSTRACT
Newborns and young infants suffer increased infectious morbidity and mortality as compared to older children and adults. Morbidity and mortality due to infection are highest during the first weeks of life, decreasing over several years. Furthermore, most vaccines are not administered around birth, but over the first few years of life. A more complete understanding of the ontogeny of the immune system over the first years of life is thus urgently needed. Here, we applied the most comprehensive analysis focused on the innate immune response following TLR stimulation over the first 2 years of life in the largest such longitudinal cohort studied to-date (35 subjects). We found that innate TLR responses (i) known to support Th17 adaptive immune responses (IL-23, IL-6) peaked around birth and declined over the following 2 years only to increase again by adulthood; (ii) potentially supporting antiviral defense (IFN-α) reached adult level function by 1 year of age; (iii) known to support Th1 type immunity (IL-12p70, IFN-γ) slowly rose from a low at birth but remained far below adult responses even at 2 years of age; (iv) inducing IL-10 production steadily declined from a high around birth to adult levels by 1 or 2 years of age, and; (v) leading to production of TNF-α or IL-1ß varied by stimuli. Our data contradict the notion of a linear progression from an 'immature' neonatal to a 'mature' adult pattern, but instead indicate the existence of qualitative and quantitative age-specific changes in innate immune reactivity in response to TLR stimulation.
Subject(s)
Cytokines/immunology , Leukocytes, Mononuclear/immunology , Toll-Like Receptors/immunology , Adult , Age Factors , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Child, Preschool , Cohort Studies , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Humans , Infant , Infant, Newborn , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lipopeptides/pharmacology , Lipopolysaccharides/pharmacology , Middle Aged , Oligodeoxyribonucleotides/pharmacology , Toll-Like Receptor 2/agonists , Toll-Like Receptor 2/immunology , Toll-Like Receptor 4/agonists , Toll-Like Receptor 4/immunology , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/immunology , Toll-Like Receptor 8/agonists , Toll-Like Receptor 8/immunology , Toll-Like Receptor 9/agonists , Toll-Like Receptor 9/immunology , Toll-Like Receptors/agonists , Young AdultABSTRACT
The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1(+)) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1(+) plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens.
Subject(s)
Antibodies, Neutralizing/immunology , Gene Products, env/immunology , HIV-1/immunology , Cell Line , HIV Infections/blood , HIV Infections/virology , HIV-1/classification , HIV-1/genetics , HumansABSTRACT
The human neonate and infant are unduly susceptible to infection with a wide variety of microbes. This susceptibility is thought to reflect differences from adults in innate and adaptive immunity, but the nature of these differences is incompletely characterized. The innate immune response directs the subsequent adaptive immune response after integrating information from TLRs and other environmental sensors. We set out to provide a comprehensive analysis defining differences in response to TLR ligation between human neonates and adults. In response to most TLR ligands, neonatal innate immune cells, including monocytes and conventional and plasmacytoid dendritic cells produced less IL-12p70 and IFN-alpha (and consequently induced less IFN-gamma), moderately less TNF-alpha, but as much or even more IL-1beta, IL-6, IL-23, and IL-10 than adult cells. At the single-cell level, neonatal innate cells generally were less capable of producing multiple cytokines simultaneously, i.e., were less polyfunctional. Overall, our data suggest a robust if not enhanced capacity of the neonate vs the adult white-blood cell TLR-mediated response to support Th17- and Th2-type immunity, which promotes defense against extracellular pathogens, but a reduced capacity to support Th1-type responses, which promote defense against intracellular pathogens.
Subject(s)
Cytokines/biosynthesis , Immunity, Innate/immunology , Infant, Newborn/immunology , Toll-Like Receptors/immunology , Adult , Cytokines/immunology , Dendritic Cells/immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Infant , Monocytes/immunologyABSTRACT
Identifying the viral epitopes targeted by broad neutralizing antibodies (NAbs) that sometimes develop in human immunodeficiency virus type 1 (HIV-1)-infected subjects should assist in the design of vaccines to elicit similar responses. Here, we investigated the activities of a panel of 24 broadly neutralizing plasmas from subtype B- and C-infected donors using a series of complementary mapping methods, focusing mostly on JR-FL as a prototype subtype B primary isolate. Adsorption with gp120 immobilized on beads revealed that an often large but variable fraction of plasma neutralization was directed to gp120 and that in some cases, neutralization was largely mediated by CD4 binding site (CD4bs) Abs. The results of a native polyacrylamide gel electrophoresis assay using JR-FL trimers further suggested that half of the subtype B and a smaller fraction of subtype C plasmas contained a significant proportion of NAbs directed to the CD4bs. Anti-gp41 neutralizing activity was detected in several plasmas of both subtypes, but in all but one case, constituted only a minor fraction of the overall neutralization activity. Assessment of the activities of the subtype B plasmas against chimeric HIV-2 viruses bearing various fragments of the membrane proximal external region (MPER) of HIV-1 gp41 revealed mixed patterns, implying that MPER neutralization was not dominated by any single specificity akin to known MPER-specific monoclonal Abs. V3 and 2G12-like NAbs appeared to make little or no contribution to JR-FL neutralization titers. Overall, we observed significant titers of anti-CD4bs NAbs in several plasmas, but approximately two-thirds of the neutralizing activity remained undefined, suggesting the existence of NAbs with specificities unlike any characterized to date.
Subject(s)
Acquired Immunodeficiency Syndrome/immunology , Antibody Specificity , HIV Antibodies/blood , HIV-1/classification , HIV-1/immunology , Animals , CD4 Antigens/physiology , Chronic Disease , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Guinea Pigs , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp41/immunology , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin G/classification , Neutralization Tests , Peptide Fragments/immunology , Receptors, CCR5/physiology , env Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
An automated method for counting spot-forming units in the ELISpot assay is described that uses a statistical model fit to training data that is based on counts from one or more experts. The method adapts to variable background intensities and provides considerable flexibility with respect to what image features can be used to model expert counts. Point estimates of spot counts are produced together with intervals that reflect the degree of uncertainty in the count. Finally, the approach is completely transparent and "open source" in contrast to methods embedded in current commercial software. An illustrative application to data from a study of the reactivity of T-cells from healthy human subjects to a pool of immunodominant peptides from CMV, EBV and flu is presented.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Models, Statistical , T-Lymphocytes/immunology , Viral Proteins/immunology , Algorithms , Humans , Image Interpretation, Computer-Assisted/methods , T-Lymphocytes/cytology , T-Lymphocytes/virology , Viral Proteins/analysisABSTRACT
Dozens of human immunodeficiency virus-type 1 (HIV-1) vaccine candidates specifically designed to elicit cytotoxic T-lymphocyte (CTL) responses have entered the pipeline of clinical trials. Evaluating the immunogenicity and potential efficacy of these HIV-1 vaccine candidates is challenging in the face of the extensive viral genetic diversity of circulating strains. Standardized peptide reagents to define the magnitude and potential breadth of the T-cell response, especially to circulating strains of HIV-1, are needed. For this purpose we developed a biometric approach based on T-cell recognition pattern for defining standardized reagents. Circulating strains in the Los Alamos database were evaluated and standardized algorithms to define all potential T-cell epitopes (PTEs) were generated. While many unique PTEs could be identified, a finite number based upon prevalence of circulating strains in the database, which we define as vaccine-important PTEs (VIPs), were used to select a common standardized panel of HIV-1 peptides for CTL-based vaccine evaluation. The usability of PTE peptide set was manifested by detection of Nef-specific CTL responses in HIV-1 subtype B infections.