Figure 1.
Cartoon depicting the HIV p55gag protein and its proteolytic cleavage products p17gag, p24gag and the C-terminal p2, p7, p1 and p6 proteins. The 7 highly conserved elements (CE) identified within p24Gag are indicated. The two p24CE constructs differ by 1 ‘toggle’ AA per CE, indicated by asterisks. The 7 CE were connected via linkers and the p24CE proteins were expressed from the CMV promoter using the mammalian expression vector pCMV.kan [18].
Figure 2.
Induction of humoral immune responses upon p24CE DNA vaccination in macaques.
(A) Cartoon depicts the vaccination schedule. Each macaque received 2 vaccinations with a mixture of p24CE1 and p24CE2 DNAs or p55gag DNA at week 0 and 8. Plasma samples were analyzed for bAb to Gag at the time of each vaccination and at the additional indicated time points. (B) Plasma bAb to HIV-1 p24Gag measured by ELISA. Mean and standard error of the reciprocal Gag antibody endpoint titers (in log10) of 10 macaques vaccinated with p24CE DNA mixture and 4 macaques which received the p55gag DNA are shown. (C, D) Western immunoblot analysis was used to identify binding antibodies to HIV-1 p24Gag and the p24CE proteins present in plasma collected after the last vaccination. The membranes contain the soluble processed clade B p24gag protein (lane 1), p24CE1 (lane 2) and p24CE2 (lane 3) protein and were probed with plasma collected after the last vaccination. The data from 2 representative macaques from each vaccine group are shown: (C) p24CE DNA vaccinated animals M437 (plasma dilution 1:2000) and P314 (plasma dilution 1:1000) and (D) p55gag DNA vaccinated animals M114 and M121 (plasma dilution 1:2000 for both). The positions of p24gag and p24CE proteins are indicated.
Figure 3.
Pepscan analysis of humoral responses induced upon p24CE and p55gag DNA vaccination in macaques.
All macaques vaccinated with (A) p24CE DNA (N = 10) and (B) p55gag DNA (N = 4) were subjected to Pepscan analysis against 58 peptides (15-mers overlapping by 11 AA) spanning p24gag. Peptides corresponding to CE1 to CE7 are indicated and the peptides are listed below as detailed in Table 1. OD450 is reported after subtracting the mean OD450 of the values obtained from negative samples plus 3 SD.
Table 1.
Peptide list and coverage of CE.
Table 2.
Responses of individual CE to cellular an humoral immune responses.
Figure 4.
HIV-1 p55gag DNA vaccination did not prime de novo CE humoral responses but increased pre-existing CE-specific responses.
(A) Cartoon depicts the vaccination schedule. After receiving p24CE or p55gag DNA as priming vaccination (see Figure 2) the animals received a heterologous boost vaccination (vaccination 3) with p55gag DNA or p24CE DNA, respectively. (B) Comparison of plasma bAb to HIV-1 p24gag ELISA titers at vaccination 3 and 2 weeks later. The reciprocal Gag antibody endpoint titers (in log10) from individual macaques vaccinated with the p24CE DNA mixture following by a p55Gag DNA boost (N = 10; left panel) and macaques vaccinated with p55Gag DNA followed by a p24CE DNA boost (N = 4; right panel) are shown. Data from day of 3rd vaccination are from Fig. 2B. (C, D) Western immunoblot analysis was used to test macaque plasma for binding antibodies before (after vaccination 2) and after the boost (after vaccination 3). The membranes contain the soluble processed p24gag (lane 1), p24CE1 (lane 2) or p24CE2 (lane 3) proteins and were probed with plasma collected 2 weeks after vaccinations 2 and 3. Data from 2 representative macaques are shown from each vaccine group. The membranes were probed with plasma from (C) macaques (L862, M166), which received the p24CE prime-p55gag DNA boost (plasma dilution 1:2000) and from (D) macaques (P574, R288), which received the p55gag prime-p24CE DNA boost regimen (plasma dilution 1:500).
Figure 5.
Pepscan analysis of humoral responses induced upon heterologous prime-boost regimens of macaques primed with p24CE priming boosted p55gag DNA boost vaccination.
Plasma collected after the prime and after the boost vaccinations were subjected to Pepscan analysis against the 58 overlapping peptides described in Figure 3. Peptides corresponding to CE1 to CE7 are indicated and the peptides are listed below as detailed in Table 1. Samples were analyzed after the priming (data from Figure 3A, left panels) and boost vaccination (right panels).
Figure 6.
Pepscan analysis of humoral responses induced upon heterologous prime-boost regimens of macaques primed with p55gag DNA and boosted with p24CE DNA.
Plasma collected after the prime (left panels) and after the boost vaccinations (right panels) were subjected to Pepscan analysis as described in Figures 3 and 5.