Protective HLA Alleles Recruit Biased and Largely Similar Antigen-Specific T Cell Repertoires across Different Outcomes in HIV Infection.

CD8+ T cells play an important role in the control of untreated HIV infection. Several studies have suggested a decisive role of TCRs involved in anti-HIV immunity. HLA-B*27 and B*57 are often associated with a delayed HIV disease progression, but the exact correlates that provide superior immunity against HIV are not known. To investigate if the T cell repertoire underlies the protective effect in disease outcome in HLA-B*27 and B*57+ individuals, we analyzed Ag-specific TCR profiles from progressors (n = 13) and slow progressors (n = 11) expressing either B*27 or B*57. Our data showed no differences in TCR diversity between progressors and slow progressors. Both alleles recruit biased T cell repertoires (i.e., TCR populations skewed toward specific TRBV families or CDR3 regions). This bias was unrelated to disease progression and was remarkably profound for HLA-B*57, in which TRBV family usage and CDR3 sequences were shared to some extent even between epitopes. Conclusively, these data suggest that the T cell repertoires recruited by protective HLA alleles are highly similar between progressors and slow progressors in terms of TCR diversity, TCR usage, and cross-reactivity.

Rapid reconstitution of CD4 T cells and NK cells protects against CMV-reactivation after allogeneic stem cell transplantation.

BACKGROUND: Epstein-Barr virus and Cytomegalovirus reactivations frequently occur after allogeneic stem cell transplantation (SCT). METHODS: Here we investigated the role of immune cell reconstitution in the onset and subsequent severity of EBV- and CMV-reactivation. To this end, 116 patients were prospectively sampled for absolute T cell (CD4 and CD8), B-cell (CD19) and NK-cell (CD16 and CD56) numbers weekly post-SCT during the first 3 months and thereafter monthly until 6 months post-SCT. Viral load was monitored in parallel. RESULTS: In contrast to the general belief, we found that early T-cell reconstitution does not play a role in the onset of viral reactivation. CMV reactivation in the first 7 weeks after SCT however resulted in higher absolute CD8(+) T-cell numbers 6 months post-SCT in patients with high-level reactivation, many of which were CMV-specific. Interestingly, rapid reconstitution of CD4(+) T-cells, as well as NK cells and the presence of donor KIR3DL1, are associated with the absence of CMV-reactivation after SCT, suggestive of a protective role of these cells. In contrast, EBV-reactivations were not affected in any way by the level of immune reconstitution after SCT. CONCLUSION: In conclusion, these data suggest that CD4(+) T-cells and NK cells, rather than CD8(+) T-cells, are associated with protection against CMV-reactivation.

IgG-mediated anaphylaxis to a synthetic long peptide vaccine containing a B cell epitope can be avoided by slow-release formulation.

Synthetic long peptides (SLP) are a promising vaccine modality to induce therapeutic T cell responses in patients with chronic infections and tumors. We studied different vaccine formulations in mice using SLP derived from carcinoembryonic Ag. We discovered that one of the SLP contains a linear Ab epitope in combination with a CD4 epitope. Repeated vaccination with this carcinoembryonic Ag SLP in mice shows improved T cell responses and simultaneously induced high titers of peptide-specific Abs. These Abs resulted in unexpected anaphylaxis after a third or subsequent vaccinations with the SLP when formulated in saline. Administration of low SLP doses in the slow-release vehicle IFA prevented the anaphylaxis after repeated vaccination. This study underscores both the immunogenicity of SLP vaccination, for inducing T cell as well as B cell responses, and the necessity of safe administration routes.

Dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and T-cell activation.

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T-cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre-)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte-derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4⁺ and CD8⁺ T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasome- and transporter associated with Ag processing-dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8⁺ T-cell activation.

Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C.

Based on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world.

Experience with synthetic vaccines for cancer and persistent virus infections in nonhuman primates and patients.

Synthetic vaccines, in particular long synthetic peptides of approximately 25-50 amino acids in length, are attractive for HIV vaccine development and for induction of therapeutic immune responses in patients with (pre-)malignant disorders. In the case of preventive vaccine development against HIV, no major success has been achieved, but the possibilities are by no means exhausted. A long peptide vaccine consisting of 13 overlapping peptides, which together cover the entire length of the two oncogenic proteins E6 and E7 of high-risk human papilloma virus type 16 (HPV16), caused complete regression of all lesions and eradication of virus in 9 out of 20 women with high-grade vulvar intraepithelial neoplasia, a therapy-resistant preneoplastic disorder. The nature and strength of the vaccine-prompted T cell responses were significantly correlated with the clinical response. Synthetic peptide vaccines are attractive, because they allow rational improvement of vaccine design and detailed pharmacokinetic and pharmacodynamic studies not possible with conventional vaccines. Improvements are possible by addition or conjugation of adjuvants, notably TLR ligands, to the synthetic peptides.

Prime-boost regimens with adjuvanted synthetic long peptides elicit T cells and antibodies to conserved regions of HIV-1 in macaques.

OBJECTIVES: Administration of synthetic long peptides (SLPs) derived from human papillomavirus to cervical cancer patients resulted in clinical benefit correlated with expansions of tumour-specific T cells. Because vaginal mucosa is an important port of entry for HIV-1, we have explored SLP for HIV-1 vaccination. Using immunogen HIVconsv derived from the conserved regions of HIV-1, we previously showed in rhesus macaques that SLP.HIVconsv delivered as a boost increased the breath of T-cell specificities elicited by single-gene vaccines. Here, we compared and characterized the use of electroporated pSG2.HIVconsv DNA (D) and imiquimod/montanide-adjuvanted SLP.HIVconsv (S) as priming vaccines for boosting with attenuated chimpanzee adenovirus ChAdV63.HIVconsv (C) and modified vaccinia virus Ankara MVA.HIVconsv (M). DESIGN: Prime-boost regimens of DDDCMS, DSSCMS and SSSCMS in rhesus macaques. METHODS: Animals' blood was analysed regularly throughout the vaccination for HIV-1-specific T-cell and antibody responses. RESULTS: We found that electroporation spares DNA dose, both SLP.HIVconsv and pSG2.HIVconsv DNA primed weakly HIVconsv-specific T cells, regimen DDDCM induced the highest frequencies of oligofunctional, proliferating CD4(+) and CD8(+) T cells, and a subsequent SLP.HIVconsv boost expanded primarily CD4(+) cells. DSS was the most efficient regimen inducing antibodies binding to regions of trimeric HIV-1 Env, which are highly conserved among the four major global clades, although no unequivocal neutralizing activity was detected. CONCLUSION: The present results encourage evaluation of the SLP.HIVconsv vaccine modality in human volunteers along the currently trialled pSG2.HIVconsv DNA, ChAdV63.HIVconsv and MVA.HIVconsv vaccines. These results are discussed in the context of the RV144 trial outcome.

Improved innate and adaptive immunostimulation by genetically modified HIV-1 protein expressing NYVAC vectors.

Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.

Long peptides induce polyfunctional T cells against conserved regions of HIV-1 with superior breadth to single-gene vaccines in macaques.

A novel T-cell vaccine strategy designed to deal with the enormity of HIV-1 variation is described and tested for the first time in macaques to inform and complement approaching clinical trials. T-cell immunogen HIVconsv, which directs vaccine-induced responses to the most conserved regions of the HIV-1, proteome and thus both targets diverse clades in the population and reduces the chance of escape in infected individuals, was delivered using six different vaccine modalities: plasmid DNA (D), attenuated human (A) and chimpanzee (C) adenoviruses, modified vaccinia virus Ankara (M), synthetic long peptides, and Semliki Forest virus replicons. We confirmed that the initial DDDAM regimen, which mimics one of the clinical schedules (DDDCM), is highly immunogenic in macaques. Furthermore, adjuvanted synthetic long peptides divided into sub-pools and delivered into anatomically separate sites induced T-cell responses that were markedly broader than those elicited by traditional single-open-reading-frame genetic vaccines and increased by 30% the overall response magnitude compared with DDDAM. Thus, by improving both the HIV-1-derived immunogen and vector regimen/delivery, this approach could induce stronger, broader, and theoretically more protective T-cell responses than vaccines previously used in humans.

Susceptibility of recently transmitted subtype B human immunodeficiency virus type 1 variants to broadly neutralizing antibodies.

The ability of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) specific human monoclonal antibodies (MAbs) b12, 2G12, 2F5, and 4E10 to neutralize recently transmitted viruses has not yet been explored in detail. We investigated the neutralization sensitivity of subtype B HIV-1 variants obtained from four primary HIV infection cases and six transmission couples (four homosexual and two parenteral) to these MAbs. Sexually transmitted HIV-1 variants isolated within the first 2 months after seroconversion were generally sensitive to 2F5, moderately resistant to 4E10 and b12, and initially resistant but later more sensitive to 2G12 neutralization. In the four homosexual transmission couples, MAb neutralization sensitivity of HIV in recipients did not correlate with the MAb neutralization sensitivity of HIV from their source partners, whereas the neutralization sensitivity of donor and recipient viruses involved in parenteral transmission was more similar. For a fraction (11%) of the HIV-1 variants analyzed here, neutralization by 2G12 could not be predicted by the presence of N-linked glycosylation sites previously described to be involved in 2G12 binding. Resistance to 2F5 and 4E10 neutralization did also not correlate with mutations in the respective core epitopes. Overall, we observed that the neutralization resistance of recently transmitted subtype B HIV-1 variants was relatively high. Although 8 of 10 patients had viruses that were sensitive to neutralization by at least one of the four broadly neutralizing antibodies studied, 4 of 10 patients harbored at least one virus variant that seemed resistant to all four antibodies. Our results suggest that vaccine antigens that only elicit antibodies equivalent to b12, 2G12, 2F5, and 4E10 may not be sufficient to protect against all contemporary HIV-1 variants and that additional cross-neutralizing specificities need to be sought.

Escape of human immunodeficiency virus type 1 from broadly neutralizing antibodies is not associated with a reduction of viral replicative capacity in vitro.

Although the majority of primary HIV-1 variants can be neutralized by broadly neutralizing antibodies such as b12, 2G12, 2F5 and 4E10, resistance to these antibodies has been reported as well. The ability of the broadly neutralizing antibodies to inhibit a variety of viruses suggests that their epitopes are conserved and escape from these antibodies may thus come at a cost to viral fitness. Here we demonstrate that resistance to broadly neutralizing antibodies was in general not associated with a reduced replicative capacity of the virus in vitro. This indicates that loss of replicative capacity due to escape from broadly neutralizing antibodies may be limited.

T cell line passage can select for pre-existing neutralization-sensitive variants from the quasispecies of primary human immunodeficiency virus type-1 isolates.

Primary human immunodeficiency type 1 viruses (HIV-1) resist antibody neutralization but become sensitive after passage through T cell lines. We and others previously reported an increased neutralization sensitivity of HIV-1 after prolonged culture on primary peripheral blood mononuclear cells (PBMC). Hence we hypothesized that adaptation to growth in T cell lines is in fact selection of a pre-existing neutralization-sensitive HIV-1 variant from the quasispecies in the PBMC culture. Indeed, increased neutralization sensitivity was associated with largely identical synonymous and non-synonymous mutations between progeny of parallel H9 passages from the same split inoculum from 2 of 3 viruses. H9 T cell line adaptation of molecular cloned HIV-1 was less successful and associated with only a few de novo mutations that varied between parallel H9-adapted progeny from the same split inoculum. We conclude that T cell line adaptation of HIV-1 can indeed select for a pre-existing variant but that this most likely depends on the viral diversity in the inoculum.

Increased neutralization sensitivity of recently emerged CXCR4-using human immunodeficiency virus type 1 strains compared to coexisting CCR5-using variants from the same patient.

CXCR4-using (X4) human immunodeficiency virus type 1 (HIV-1) variants evolve from CCR5-using (R5) variants relatively late in the natural course of infection in 50% of HIV-1 subtype B-infected individuals and subsequently coexist with R5 HIV-1 variants. This relatively late appearance of X4 HIV-1 variants is poorly understood. Here we tested the neutralization sensitivity for soluble CD4 (sCD4) and the broadly neutralizing antibodies IgG1b12, 2F5, 4E10, and 2G12 of multiple coexisting clonal R5 and (R5)X4 (combined term for monotropic X4 and dualtropic R5X4 viruses) HIV-1 variants that were obtained at two time points after the first appearance of X4 variants in five participants of the Amsterdam Cohort Studies on HIV-1 infection and AIDS. Recently emerged (R5)X4 viruses were significantly more sensitive to neutralization by the CD4-binding-site-directed agents sCD4 and IgG1b12 than their coexisting R5 viruses. This difference was less pronounced at the later time point. Early (R5)X4 variants from two out of four patients were also highly sensitive to neutralization by autologous serum (50% inhibition at serum dilutions of >200). Late (R5)X4 viruses from these two patients were neutralized at lower serum dilutions, which suggested escape of X4 variants from humoral immunity. Autologous neutralization of coexisting R5 and (R5)X4 variants was not observed in the other patients. In conclusion, the increased neutralization sensitivity of HIV-1 variants during the transition from CCR5 usage to CXCR4 usage may imply an inhibitory role for humoral immunity in HIV-1 phenotype evolution in some patients, thus potentially contributing to the late emergence of X4 variants.