shows the immunization schedule. A total of six macaques were used in this study of which three (51M, 978L, 980L) were immunized with a mixture of six plasmids encoding the codon-optimized env gp120 genes and a gag p55 gene () by i.m. route and another three (991L, 997L, 998L) received the same DNA mixture by i.d. route. During the primary phase of immunization animals were vaccinated with the multivalent DNA immunogens on weeks 0, 6, 12 and 18 followed by boosting with homologous Env protein in QS-21 adjuvant on weeks 24 and 32. After detection of strong antibody and cell-mediated responses elicited from this primary phase of immunization, animals were subsequently rested for 33 weeks and then boosted with a single multivalent DNA immunization on week 65 followed by two protein boosts on weeks 76 and 93 (secondary immunization phase). Animals were challenged with SHIVBa-L via the rectal route on week 95, 2 weeks after the last protein boost.
Fig. 1 Immunization schedule. Arrows indicate times of vaccination. Six macaques were divided into two groups. Three animals were immunized with polyvalent DNA by the i.m. route and the other three by the i.d. route followed by protein immunization intramuscularly. (more ...)
Antibody response in macaques following the primary phase of immunization of DNA prime/protein boost vaccine
Antibody titers to all five Env gp120 proteins following each immunization of DNA and protein were assayed and the results are shown in . The first DNA immunization elicited a very low level of antibody response to Env proteins (data not shown). DNA delivered via the i.m. route elicited slightly higher antibody responses to at least three of five Env gp120 proteins and the antibody titers increased progressively following each DNA immunization. However, boosting of DNA-primed animals with gp120 proteins enhanced antibody titers markedly in both groups of animals to a comparable level. Titers of anti-Gag antibodies were present but at low levels in each group during both DNA and protein immunization phase (data not shown).
Fig. 2 Anti-gp120 titers in macaques immunized with polyvalent DNA and gp120 protein during the primary immunization phase. Anti-Env titers in the sera of macaques immunized with DNA and gp120 proteins were assayed by ELISA against A, B715, Ba-L, C and E gp120 (more ...)
Sera harvested 2 weeks after the fourth DNA and the single protein boost were assayed for neutralization of SHIVBa-L and a few HIV-1 isolates homologous to the DNA vaccines. Sera after DNA immunizations did not neutralize any of the viral isolates tested (data not shown). However, sera collected after a single protein boost elicited neutralizing antibodies to the homologous isolates (). These sera were then tested for neutralization of heterologous isolates including HIV-1MN and a few primary HIV-1 isolates from different clades from which the immunogens were derived. As shown in , HIV-1MN and primary isolates SF162 and Bx08 were clearly neutralized by the sera of all immunized animals with animal 998L demonstrating the highest neutralizing antibody titers among all the animals. A few sera were able to neutralize clade B isolates ADA and JRCSF. In addition, clade C isolate TV1 was neutralized by three sera whereas clade A isolate DJ263 was neutralized by sera from four animals. No sera were able to neutralize clade E isolate CM235.
Neutralization of homologous HIV-1 and SHIVBa-L isolates by the sera from macaques immunized with polyvalent DNA prime/protein boost vaccine
Neutralization of heterologous HIV-1 isolates by sera from macaques immunized with polyvalent DNA/protein vaccine
Antibody response during the secondary immunization phase
Ideally an effective prophylactic vaccine should maintain high-titered antibodies in serum. Alternatively, if decay in antibody titer is observed, additional immunization with the vaccine should boost the titers markedly. Hence it was of interest to determine the decay of the anti-gp120 antibody response after primary immunization phase and whether such antibody response could be reboosted with the same polyvalent DNA and protein immunizations. After 33 weeks of rest following the second protein boost (on week 32), animals were reboosted with DNA on week 65 and with proteins on weeks 76 and 93. As shown in , anti-gp120 titers against Ba-L gp120 dropped slowly with time following the second protein boost on week 32, but immunization with a single DNA and two protein boosts increased anti-gp120 titers slightly higher than that observed during the primary immunization phase. Similar results were observed when anti-gp120 titers were assessed against other gp120 proteins included in the immunogen () (data not shown). These results clearly demonstrate that although antibodies elicited during the primary immunization phase decayed, such responses could be easily boosted back to the original level with limited immunizations with DNA and protein vaccines.
Fig. 3 Anti-gp120 titers in macaques immunized with polyvalent DNA and gp120 protein during the secondary immunization phase. Anti-Ba-L gp120 titers were measured in the immunized macaques by ELISA during both primary and secondary immunization phases. After (more ...)
HIV-1 Gag cellular immune responses elicited following DNA immunizations
HIV-1 Gag antigen was included in the DNA vaccine phase of this multivalent DNA prime/protein boost vaccine to increase the breadth of cellular immune response elicited by the Env-based DNA and protein immunogens. In order to demonstrate that Gag-specific CMI responses were elicited following DNA immunization, PBMC from immunized macaques were isolated 2 weeks after the fourth DNA immunization (week 20) and the frequencies of Gag-specific lymphocytes were measured by IFN-γ ELISPOT assay, using HIV-1 MN Gag peptide pools (15-mer with 11 amino acid overlap, 12 peptides in each pool). As shown in , all three animals in the i.m. immunization group and two of three animals in i.d. immunization group demonstrated CMI responses against one or more of the 10 Gag peptide pools. The numbers of net IFN-γ spot-forming cells ranged from 12 to 300 per million PBMCs for each peptide pool.
Fig. 4 Gag peptide-specific cellular immune responses after the fourth DNA immunization. The numbers of IFN-γ spot-forming cells per million PBMC were measured by ELISPOT assays as described in the Materials and Methods section. Macaques received HIV-1 (more ...)
Virological status after rectal challenge with SHIVBa-L
As the immunogens used in the vaccine originated from R5 SHIV isolates, immunized animals were challenged rectally with an R5 tropic SHIVBa-L isolate encoding HIV-1Ba-L Env homologous to one of the immunogens. As shown in , this challenge virus is a neutralization-sensitive isolate. Plasma viral RNA loads in the naïve and immunized animals over time are shown in . As expected, all six naïve animals were infected following rectal challenge with SHIVBa-L (). Only one macaque (980L) immunized with DNA by the i.m. route was protected from challenge. The second primate, 51M, had significantly lower plasma viremia detected only on days 28 and 35 compared with the rest of the infected animals (). Overall, RNA load of the i.m. group was significantly lower than the naïve group (P < 0.033), whereas the difference in RNA loads between i.d. and naïve groups was approaching significance (P < 0.058). When RNA load of both i.m. and i.d. groups were combined, the reduction in viral load was significant (P < 0.011), and when the protected macaque (980L) was removed from the comparison, the difference was still significant (P < 0.024) (). The infectious status of each animal was also confirmed by qualitative detection of SIV proviral DNA from PBMC collected after 120 days following challenge by PCR assay with primers specific to gag gene. Macaque 980L with no detectable plasma viremia was also negative for proviral DNA in PBMC (data not shown). Interestingly, macaque 51M with detectable plasma viremia on days 28 and 35 was also shown to be negative for proviral DNA in PBMC (data not shown).
Fig. 5 Plasma viral RNA load in macaques following SHIVBa-L challenge. Plasma viral RNA (copies/100 μl) was determined by nucleic-acid sequence-based amplification assay (NASBA). RNA loads of individual animals from naïve (A) and immunized (B) (more ...)
Post challenge immune response
Both humoral and cellular immune responses were evaluated in immunized macaques following viral challenge and the results are shown in . As expected, all the immunized animals had high-titered anti-gp120 antibodies on the day of challenge which stabilized three weeks post-infection (data not shown). Neutralizing antibodies to the challenge virus (SHIVBa-L) were detected on the day of challenge in all macaques with the highest titer observed in animal 998L (). Neutralization titers remained steady up to 18 days post-challenge and increased thereafter (). In contrast, neutralizing antibody to SHIVBa-L was detected in naïve animals only after 4 weeks (data not shown). Frequency of Ba-L Env-specific lymphocytes was also evaluated in the challenged animals by IFN-γ ELISPOT assay. All the immunized macaques had a significant level of Env-specific CMI response on the day of challenge (). Although macaque 998L had significantly higher neutralizing antibody and CMI responses compared with other animals, such responses failed to protect this animal from infection.
Fig. 6 Post-challenge immune response in challenged macaques. (A) Neutralizing antibody titers in sera collected on the day of challenge and at multiple points post-challenge were determined against SHIVBa-L in U373 cells as described in the Materials and Methods (more ...)