We characterized anti-orthopoxvirus NAb responses elicited by MVA vaccination at doses from 107 to 109 pfu in subjects who were inoculated with MVA or MVA followed by FPV. Neutralizing antibody responses against MVA and VACV were clearly generated by the 108 and 109 doses of MVA, but only a minority of recipients in the 107 dose group seroconverted against VACV. Interestingly, a single administration of MVA in the highest dose groups (109 MVA(2)/FPV(3) or MVA(5)) elicited detectable anti-MVA and anti-VACV NAb titres in most subjects at day 14 compared to a 50% response rate to MVA and 25% response rate to VACV in the 108 MVA(2)/FPV(3) group after a single injection. These titres substantially increased by day 42 and all subjects in the 109 MVA(2)/FPV(3) and MVA(5) groups seroconverted.
Further inoculations of MVA did not lead to boosting in MVA or VACV NAb titres, but did lead to a slower decline in titres compared with the 109 MVA(2)/FPV(3) group. By day 394, 97% of subjects in the 109 MVA(5) group still had detectable anti-MVA NAb responses compared with 81% in the 109 MVA(2)/FPV(3) arm. For VACV-specific neutralization, the difference was more pronounced: 64% for the 109 MVA(5) group versus 30% in the 109 MVA(2)/FPV(3) arm. This suggests that two doses of MVA are optimal, at least for eliciting anti-orthopoxvirus responses, although the utility of booster doses on a longer time scale are not known.
MVA has most often been administered twice at a one month interval [6
]. One study using MVA-TBC (Therion), similar to the strain used in HVTN 055 but at a dose of 1 × 106
pfu, inoculated volunteers up to three times and found little difference in orthopoxvirus-specific NAb titres, CD4+ T cell responses, or CD8+ T cell responses between subjects vaccinated with MVA once, twice, or thrice prior to Dryvax challenge [15
]. In HVTN 065, rMVA with HIV-1 inserts was given twice following a DNA priming vaccine versus thrice without a prime. Recipients in the DNA prime/rMVA boost arm had significantly lower VACV-specific T cell responses after the first rMVA boost than subjects who received rMVA alone, but this inhibition was overcome by the second dose of rMVA [16
]. In two studies using MVA to deliver melanoma-derived CD8+ T cell epitopes, the rMVA-Mel3 vaccine was given up to four times at doses ranging from 107
pfu to patients with melanoma [17
]. Anti-MVA binding antibodies and MVA-specific T cells were elicited but detailed kinetics for the humoral responses were not described [17
]. MVA-specific T cell responses were generally maximal after two injections, although two of five patients had increased VACV epitope-specific CD8+ T cell responses after all four inoculations [19
Although we did not see evidence of boosting in NAb titres after the second immunization, there was boosting of insert-specific CD8+ T cell responses in the rMVA-HIV(2)/rFPV-HIV(3) groups, particularly at the highest dose [4
]. In contrast, heterologous boosting did not increase anti-Env antibody responses which were highest in the 109
rMVA-HIV(5) group [4
]. We explored the relationship between anti-vector and anti-insert responses and we found minimal interaction between the two. High MVA or VACV NAb titres were not associated with lower T cell responses to the HIV inserts, suggesting that anti-vector responses do not interfere with development of responses to the immunogen. There was some evidence of a positive association between VACV neutralization and CD8+ T cell responses among vaccinees who received rMVA-HIV(2)/rFPV-HIV(3), although this association was not seen with MVA neutralization. There was no association between MVA neutralization and ELISA responses against gp120 or p24, but vaccinees receiving rMVA-HIV(5) who had high VACV neutralization activity were more likely to have an antibody response to gp120.
In an analogous macaque study using rVACV, rMVA, and rFPV vectors from Therion (similar to the vectors used in HVTN 055) at a dose of 2 × 109
pfu which delivered SIV-derived inserts [20
], our group found evidence that a third dose of rMVA did elicit boosting of anti-orthopoxvirus humoral responses [7
]. In a regimen of rMVA-SIV at weeks 0, 8, 26, and 43, higher anti-VACV binding antibodies were seen at week 48 than in animals inoculated with either rMVA-SIV or rVACV-SIV followed by rFPV-SIV. Anti-vector NAb and comet neutralization were also boosted after the third dose [7
]. A fourth dose of rMVA-SIV did not lead to increases in anti-orthopoxvirus NAb titres, but peak titres were maintained for over four months [7
]. The differences in the vaccination schedule or dose of rMVA between the macaque study and HVTN 055 could explain the differences we observed in the pattern of immune responses following booster inoculation.
Our results from HVTN 055 highlight several important concepts. First, orthopoxvirus-specific NAbs are induced by rMVA inoculation in a dose-dependent fashion from 1 × 107 to 1 × 109 pfu. While a second dose boosts NAb titres, further doses do not yield higher titres but do significantly delay the decay in NAb titres over more than one year of follow-up. Furthermore, orthopoxvirus-specific NAb titres were not different between subjects administered rMVA-HIV compared with those inoculated with the empty vector, suggesting that the HIV-1-derived insert does not interfere with induction of anti-vector humoral immune responses. Finally, anti-vector NAb responses elicited by rMVA-HIV do not interfere with the development of responses directed against the HIV-1-derived insert in an orthopoxvirus-naive cohort. In fact, the associations we identified suggest that there may be a positive, albeit modest, interaction between anti-vector and anti-insert responses. This possible association may reflect underlying host genetics or adjuvant-like properties of the viral vector and should be examined in a larger cohort with detailed examination of vector-induced cellular immune responses.