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Vaccination with MV1-F4 induces strong F4- and MV-specific T cell responses in mice

The immunogenicity of MV1-F4 recombinant vaccine was first evaluated in genetically modified CD46-IFNAR mice susceptible to MV infection. Intracellular cytokine staining was detected by flow cytometry following in vitro stimulation of freshly extracted splenocytes with HIV-1 F4 peptide pools (Figure 1A and B) and empty MV (Figure 1C and D). Intracellular cytokine staining for IFNγ and IL-2 was observed in both CD4+ and CD8+ T cells from immunised animals, as compared with non-immunised control mice. The intensity of response, expressed as the percentage of single or double cytokine-positive CD4+ and CD8+ cells, was dependent on the inoculated dose with a marked increase with the highest dose (107 TCID50), resulting in strong HIV and MV responses. Single and double cytokine staining for IFNγ and IL-2 was observed in both HIV F4- and MV-specific CD4+ and CD8+ T cells. However, IFNγ was produced in a much higher amount than IL-2. The percentages of CD4+ T cell cytokine responses were at least 2 times higher than CD8+, both for HIV and MV. Altogether, this analysis shows that MV1-F4 is strongly immunogenic and elicits a high level of CD4+ and CD8+ T cell responses in CD46-IFNAR mice, supporting its further evaluation in non-human primates.


Vaccination with MV1-F4 induces polyfunctional T cell responses to HIV in macaques

Polyfunctional CD4+ and CD8+ T cell cytokine responses were detected by flow cytometry following in vitro stimulation of PBMC with HIV-1 F4 peptide pools (Figure 2). Single, dual and triple cytokine staining for TNFα, IL-2 and IFNγ by CD4+ and CD8+ T cells was observed (Figure 2A–D). The activation marker CD154, also known as CD40 ligand, was co-expressed by cytokine positive CD4+ T cells (Figure 2A and B).


Following vaccination, potent CD4+ T cell cytokine responses against HIV-1 F4 insert peptide pools were detected in macaques F52 and F53 from group A and macaques F56 and F57 from group B (Figure 3A and B). By contrast, macaques F51 and F54 from group A and F55 and F58 from group B did not demonstrate significant CD4+ T cell cytokine responses (Figure 3A and B). The CD4+ T cell response of macaque F55 was ambiguous as it was low and occurred only at a single, late time point (Figure 3B). Only one animal in group A, F52, showed any sign of a boosted CD4+ and CD8+ T cell response against F4 peptides after a second vaccination. The CD4 responses of F53 were already high and continuing to rise at the time of boosting peaking on day 42. Responses in both groups declined sharply at day 84 and could not be detected in lymphoid tissues taken at termination (Figure 3A and B). Significant CD8+ T cell cytokine responses against HIV-1 F4 insert peptide pools were detected in all animals (Figure 3C and D). However, compared with the CD4+ T cell responses against HIV-1 F4 peptides, the magnitude of CD8+ T cell responses was moderate. CD8+ T cell cytokine responses were overall greater and appeared earlier in group B than in group A, but peripheral responses in both groups declined sharply at day 84. Nonetheless, significant HIV-1 F4-specific CD8+ T cell responses could still be detected in spleen cells collected over 3 months after the last immunisation, from all macaques except F51 and F53 (Figure 3C and D).


Vaccination with MV1-F4 induces humoral responses to HIV in macaques

Macaques F51, F52, F53, F54 and F57 developed binding antibodies against HIV-1 F4 antigen as soon as 14 days after immunisation with MV1-F4 (Figure 5A). A very low F4-specific binding antibody response was detected in the F55 animal only at day 14. Seroconversion of macaque F51 was only detected after a second immunisation (Figure 5A). Titres of anti-F4 binding antibodies in macaques F52, F53 and F54 were boosted after a second immunisation, while the anti-F4 humoral responses in the F57 animal waned at day 56. Macaques F56 and F58 did not seroconvert to the F4 antigen (Figure 5A).




























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