DISCUSSION
infection
DISEASE PROGRESSION, DISEASE, INFECTION
The induction of T cell responses capable of inhibiting viral replication by vaccination is potentially an important endpoint to assess in clinical HIV vaccine trials, as this is a putative mechanism by which vaccine-induced immunogenicity may contribute to limiting viral replication following HIV-1 transmission or control systemic viral load in the context of therapeutic vaccination. Vaccines inducing functional non-neutralizing antibodies have not yet been able to demonstrate sufficient clinical efficacy in experimental trials (In this work, we demonstrate that the Ad26/Ad26 + gp140 and Ad26/MVA + gp140 vaccine regimens tested in the APPROACH clinical trial elicited CD8+ T cell responses to HIV Pol, Gag, and Env. These CD8+ T cells were able to inhibit replication of HIV in autologous CD4+ T cells, with 94% of vaccinees inhibiting at least one HIV isolate. There were no striking differences observed between the magnitudes post 3rd and post 4th vaccination visit, but overall, the Ad26/MVA + gp140 regimen appeared to induce somewhat stronger inhibitory responses than the Ad26/Ad26 + gp140 vaccine regimens at the post 3rd vaccination timepoint, whereas this varied per viral isolate post 4th vaccination. This indicates that both regimens induce immune responses that are able to inhibit viral replication. The commonality of the first two vaccinations with Ad26.Mos.HIV vaccine used in both arms may be responsible for priming a similar induction of these effects.Clustering analysis demonstrated that participants’ samples from both time points tend to give very similar results, indicating that inter-individual variability in responses to vaccination, rather than technical or biological variation in the assay, is driving the observed results. When considering the clustering of the viral isolates, this appears driven by magnitude of viral inhibition, rather than by responses to the clade of the isolates, indicating that the mosaic vaccine antigens employed here do not skew inhibitory responses to a specific clade, but rather broad viral inhibition is achieved in this panel. Although there was no strong clustering of responses by CCR5- or CXCR4-tropic viral isolates, we did observe that the three highest inhibited viral isolates were CXCR4-tropic. As the vaccine constructs are most similar to CCR5-tropic envelopes and the 3rd and 4th vaccination includes the CCR5-tropic C97ZA gp140 protein, this demonstrates effective cross-tropism responses. Although CXCR4-tropic viruses make up only approximately 5% of transmitted viruses (Of note, the strain that was autologous to the protein component of the vaccine (ZA97012) was not inhibited better than heterologous strains (7 out of 16 responders), suggesting that the observed inhibition of viral replication is induced by the vector-based components of the vaccine, rather than driven by the protein component. Mosaic antigen delivery via viral vectors will ensure intracellular expression and processing of the antigen (As it was observed that the percentage amino acid overlap between the mosaic HIV vaccine sequence and the sequences of HIV-1 isolates tested in the VIA demonstrated no significant correlations with VIA outcomes, efficient inhibition of viral replication was independent of closely matched sequences. As the mosaic antigen design ensures targeting of epitopes conferring the greatest breadth of known viral sequences, this is in line with the concept of broad T cell coverage being achieved through mosaic immunization. Alternatively, sequence overlap in a specific epitopic site, rather than overall sequence overlap, may be of more relevance for high viral inhibitory activity, as this was previously found to be linked to protection (Viral inhibition by CD8+ T cells may be mediated by a variety of different mechanisms. TCR stimulation may lead to the secretion of cytolytic molecules such as granzyme B as well as release of inflammatory cytokines such as IFN-γ and β-chemokines which may block the entry of CCR5-tropic viruses (Participants that demonstrated the broadest VIA responses showed positive correlations with ELISpot responses to a sum of Env, Gag, and Pol responses. Interestingly, they did not typically score as the highest responders to each of the Env, Gag, and Pol antigens in the ELISpot assay but rather responded strongly to only one of these proteins. The PBMC ELISpot mapping further supports that each individual demonstrates a unique pattern of targeted regions within the Env, Gag, and Pol proteins irrespective of VIA breadth. This could be an attribute of the mosaic vaccine design, as the elicited T cells would inherently have optimal coverage for any given antigenic specificity. It could be postulated that inducing an extended repertoire of T cells responsive to different regions of Env, Gag, or Pol antigens is associated with breadth of viral inhibition irrespective of their exact specificities. Indeed, breadth of antigen recognition by CD8+ T cells is inversely associated with viral load and disease progression in individuals living with HIV-1 (Besides ELISpot, breadth of HIV-1 inhibition also showed positive correlation with CD8+ T cell PFS COMPASS scores to Gag and Env peptide pools. These polyfunctionality correlations were stronger than those with IFN-γ and/or IL-2-expressing T cells. COMPASS is designed to evaluate polyfunctionality of T cells through exploring all functional T cells subsets within a multi-color flow cytometric panel (Comparisons of VIA data from the APPROACH trial to two candidate HIV-1 vaccine regimens also designed to elicit HIV-1 specific T cell responses, the Merck Ad5 and VRC DNA/Ad5 vaccines (There are some limitations to our study. First, participants with a range of quantifiable vaccine-induced IFN-γ ELISpot responses to Mosaic or PTE peptides to Env, Gag, and Pol antigens were selected for inclusion in the VIA analysis to ensure maximal likelihood of detecting a diverse range of VIA responses, which may have resulted in an estimation of the induced inhibitory responses that is not representative of the overall study population. Second, the limited number of participants included in this study precludes any generalizable conclusions from being drawn. More elaborate studies using T cells from a fully representative subset of trial participants will provide better insight into vaccine-induced inhibitory activity. Third, the VIA responses measured in this phase 2a clinical trial could not be linked to any vaccine efficacy data, so their relevance to protection from infection or disease remains to be established through comparing inhibitory responses between infected and uninfected vaccinated participants in efficacy studies.As the data presented here indicate that strong viral inhibition responses can be induced by the Ad26 mosaic-based vaccine regimen, it could be of interest to assess if induction of broad viral inhibition responses is associated with vaccine efficacy in the phase 2b and phase 3 clinical trials that tested adapted versions of this regimen and if the relationships described in this work between VIA breadth and ELISpot and ICS responses can be reproduced and recapitulated in a larger case-control data set. This would contribute to understanding a specific mechanism through which vaccine-induced cellular immune responses may limit HIV-1 replication and their relevance for the development of an efficacious HIV vaccine that may help end the HIV-1 pandemic.