An immunization protocol that focuses the immune response on the structurally-conserved V3 epitope induced Abs in rabbits that were capable of neutralizing psVs and primary isolates constructed or isolated from subjects infected with HIV subtypes A, AG, B, C, and D. These psVs and primary isolates were derived from patients who were acutely or chronically infected by heterosexual or homosexual exposure. The neutralizing activity was assessed in three different assays, run in three independent laboratories, and was shown by peptide blocking experiments to be directed against V3 epitopes. The immunization regimen that gave the most consistent and broadest responses when assessed in all three assays was that in which the animals were primed with subtype C gp120 DNA and boosted with V3B-FP; the breadth of this response was greater than that displayed by pools of human anti-V3 mAbs.
Given the variation in results achieved with different neutralization assays and the challenges of standardizing a single assay for use in several labs, the immune rabbit sera generated by the DNA prime/protein boost protocols used here were tested against three different groups of viruses and psVs in three different assays in three different labs: (a) Clonal V3 chimeric psVs carrying the V3 sequences of various subtypes presented in the context of the neutralization-sensitive SF162 backbone were tested using the CD4+CCR5+ U87 line as target cells. (b) Primary isolates grown in PBMCs were tested using the engineered CD4+CCR5+ TZM-bl line as target cells. And (c) psVs constructed with an uncloned panel of primary envelopes derived from the plasma of HIV+ individuals were tested using the CD4+CCR5+CXCR4+ U87 line as target cells. The latter assay was run on coded samples. The data from all of these assay systems were consistent in showing that priming with DNA containing a V3 sequence characterized by the GPGQ motif at the tip was a better prime than a DNA prime containing a V3 sequence carrying the GPGR motif. The immunization regimen that gave the strongest and/or broadest neutralizing Ab responses utilized the clade C gp120 DNA prime together with boosts of the V3B-FP. Various combinations of V3A-,V3B-, and/or V3C-FPs also induced cross-clade neutralizing Abs, although the level and breadth of positive results from animals receiving the combined V3-scaffold protein boosts were less reliably demonstrated in all of the neutralization assays.
The demonstration that the CQ
/B immunized group gave the highest ELISA titers and the most consistent cross-clade neutralizing Ab response reflects tenets in the classic immunologic literature relevant to the induction of cross-reactive Abs. Thus, priming with immunogen A and boosting with immunogen B is known to induce Abs reactive with both immunogens (Richards et al., 1975
). The data presented above are also consistent with previously published data that suggest that immunizing with constructs from more than one clade of HIV induces broader immune responses than immunizing with constructs from a single clade (Burke et al., 2009; Seaman et al., 2005; Wang et al., 2006; Zolla-Pazner et al., 2008
). However, the data presented indicate that some combinations, e.g., CQ
/B, are better than others. Determining which prime and boost will give the optimal qualitative response, i. e., Abs with the broadest neutralizing activity, can be address rationally and informed by both structural and immunologic studies of monoclonal Abs. However, it is noteworthy that in the experiments described above the group receiving the multivalent prime (see ) did not give as strong or broad a neutralizing response as the groups primed with the clade C gp120 alone. This quantitative effect of dose in multivalent vaccines needs further study.
The level of neutralizing Abs achieved in these experiments varied from 1:10-1:2400 depending on the assay system and the virus or psV that was employed. V3 chimeric psVs carrying the Env of the neutralization-sensitive SF162 virus were, not surprisingly, the most sensitive (). Primary isolates known to be neutralization sensitive, e.g., BX08, BZ167 and DJ263, gave the next highest titers, with titers often >1:100 ( and ). When more resistant viruses and psVs (those often referred to as “Tier 2”) were tested, detectable GMT50
values for neutralization Ab ranged from 1:10 to 1:102 ( and ). Many of the primary isolates were in early passage after isolation (NYU3738, NYU6525, NY129), and had been grown in PBMCs. Since such viruses are more resistant than psVs when tested in the TZM assay, these primary isolate neutralizing titers, which were calculated on the basis of 2-3 titration experiments each run in triplicate against a multiclade virus panel, are especially noteworthy. The titers against “Tier 2” viruses and psVs are quantitatively comparable or stronger, and qualitatively broader, than those recently reported by others (Burke et al., 2009; Law et al., 2007; Mascola et al., 2005
; Morner et al., 2009; Seaman et al., 2005; Vaine et al., 2008; Wu et al., 2006).
Protective levels of vaccine-induced serum neutralizing Abs against polio have been reported at 1:4 (Plotkin, 2008
) and ≥1:20 (Wicker et al., 2007
). Similarly, vaccine-induced serum hemagglutinin inhibitory Ab titers ≥1:40 are considered to be the protective threshold for influenza (Hannoun, Megas, and Piercy, 2004
). The relevance of these values to protective Ab titers against HIV is open to question. However, challenge with various strains of SHIV in passively immunized macaques demonstrates that serum neutralizing titers of 1:1 (neat serum) to 1:38 can protect against infection (Hessell et al., 2009
; Mascola et al., 2000
; Shibata et al., 1999
). Such low Ab titers that confer protection suggest that previous conclusions about the need for high Ab titers (Baba et al., 2000
; Parren and Burton, 2001
; Parren et al., 2001
) may be unwarranted. It is noteworthy, however, that these data are derived from experiments in which the challenging “animal infectious doses” used induced infection in most or all control animals. In contrast, infectious doses for human heterosexual transmission per contact is on the order of one in 500 (Dunkle et al., 2008; Gray et al., 2001). Given the requirements of experimental design in animal studies to insure statistically meaningful results, the infectious doses used in animal models appear to be orders of magnitude larger than those occurring in humans. Thus, the level of Abs needed to protect humans may be much less than that needed to protect animals that are infected in most experimental models, and it is probable that the level of Abs required to protect against natural human infection will not be established until protection is achieved with a vaccine for humans. Therefore, it is impossible to know whether the relatively low vaccine-induced cross-clade neutralizing Ab titers which have been reported by us and others would be protective. However, now that considerable breadth has been achieved, additional experiments using alternative adjuvants, vaccine formulations and delivery systems should significantly increase Ab levels, and thus achieve the desired goal of inducing neutralizing Abs with a breadth and potency capable of preventing infection with HIV.
While a yet-to-be-defined minimal Ab titer is of critical significance for protection against infection, induction of Abs with broad reactivity is an absolute requirement for any HIV vaccine candidate; consequently, the aim of the experiments described here was to increase the breadth of the neutralizing Ab response. One of the variables examined to accomplish this was the nature of the prime. Although the composition of gp120 molecules from clades B and C are known to differ by ~30%, one crucial difference which is especially relevant to the induction of anti-V3 Abs occurs at the tip of the V3 loop, with the GPGR motif characterizing most clade B viruses and the GPGQ “tip type” occurring in most clade C viruses. This R/Q distinction has been shown to have considerable antigenic significance in that the R/Q mutation can alter neutralization sensitivity (Zolla-Pazner et al., 2004
). Similarly the R/Q difference between B and non-B clades affects to the breadth of anti-V3 Abs produced by HIV-infected individuals (Brown et al., 2008; Gorny et al., 2004; Gorny et al., 2006; Krachmarov et al., 2006
; Krachmarov et al., 2005). This R/Q difference appears to have contributed to the broader anti-V3 Ab response induced by the clade C gp120 DNA prime. This conclusion is supported by the finding that mAbs that engage the R residue in the GPGR motif at the tip of the V3 are quite restricted in their neutralizing activity for viruses carrying the GPGR tip type (Binley et al., 2004; Stanfield et al., 2004; Zolla-Pazner et al., 2004
). In contrast, anti-V3 mAbs where the R/Q residue at the tip of V3 does not interact with the Ab surface are able to neutralize both B and no-B clade viruses ((Stanfield et al., 2006
) and unpublished data). The data suggest that the CQ
prime establishes memory for anti-V3 Abs that are independent of the V3 tip type, and that, in the context of broadly reactive anti-V3 memory cells, the V3-FP boosts activate those memory B cells that are maximally cross-reactive and capable of binding to both GPGR-containing and GPGQ-containing V3 peptides and of neutralizing viruses from many subtypes regardless of the motif at the tip of the V3 loop.
As shown in , the rabbits receiving the CQ prime and any one of the V3-FPs produced Abs that could neutralize primary isolates that were not neutralized by the anti-V3 mAbs pools. Thus, the polyclonal Abs induced with a multi-clade, V3-focused immunization protocol showed broader neutralizing activity than cocktails of various anti-V3 monoclonal Abs. In neutralization assays for polyclonal Abs against recombinant psVs constructed from the Envs of patients infected acutely or chronically with various subtypes (), the group of rabbits immunized with CQ/B gave the broadest response: 66% of the 100 serum/psV combinations were neutralized by the sera from the five animals in this group, and four of the five rabbits in this group neutralized psVs from clades B, C and D. In contrast, several of the psVs neutralized by the immune rabbit sera were neutralized poorly or were entirely resistant to anti-V3 mAbs pools or to a broadly neutralizing HIV+ human serum used as a positive control. Moreover, four of five immune sera from the CQ/B group neutralized primary isolates and psVs from subtypes A, AG, B, C and D in the various neutralization assays performed in different labs (, and ). Taking all of the data together, immunization with the CQ/B regimen was shown to be capable of inducing the most broad, cross-clade neutralizing Ab response.