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. 2012 Aug 10;30(37):5519-26.
doi: 10.1016/j.vaccine.2012.06.042. Epub 2012 Jun 27.

Motif-optimized subtype A HIV envelope-based DNA vaccines rapidly elicit neutralizing antibodies when delivered sequentially

Franco Pissani  1 , Delphine C Malherbe, Harlan Robins, Victor R DeFilippis, Byung Park, George Sellhorn, Leonidas Stamatatos, Julie Overbaugh, Nancy L Haigwood
Affiliations

Motif-optimized subtype A HIV envelope-based DNA vaccines rapidly elicit neutralizing antibodies when delivered sequentially

Franco Pissani et al. Vaccine. .

Abstract

HIV-1 infection results in the development of a diverging quasispecies unique to each infected individual. Envelope (Env)-specific neutralizing antibodies (NAbs) typically develop over months to years after infection and initially are limited to the infecting virus. In some subjects, antibody responses develop that neutralize heterologous isolates (HNAbs), a phenomenon termed broadening of the NAb response. Studies of co-crystalized antibodies and proteins have facilitated the identification of some targets of broadly neutralizing monoclonal antibodies (NmAbs) capable of neutralizing many or most heterologous viruses; however, the ontogeny of these antibodies in vivo remains elusive. We hypothesize that Env protein escape variants stimulate broad NAb development in vivo and could generate such NAbs when used as immunogens. Here we test this hypothesis in rabbits using HIV Env vaccines featuring: (1) use of individual quasispecies env variants derived from an HIV-1 subtype A-infected subject exhibiting high levels of NAbs within the first year of infection that increased and broadened with time; (2) motif optimization of envs to enhance in vivo expression of DNA formulated as vaccines; and (3) a combined DNA plus protein boosting regimen. Vaccines consisted of multiple env variants delivered sequentially and a simpler regimen that utilized only the least and most divergent clones. The simpler regimen was as effective as the more complex approach in generating modest HNAbs and was more efficient when modified, motif-optimized DNA was used in combination with trimeric gp140 protein. This is a rationally designed strategy that facilitates future vaccine design by addressing the difficult problem of generating HNAbs to HIV by empirically testing the immunogenicity of naturally occurring quasispecies env variants.

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Figures

Figure 1
Figure 1. Phylogenetic analysis of QA255-derived Env quasispecies
(A) Maximum Likelihood (ML) phylogeny illustrating the inferred relationships between env variants isolated from subject QA255 and chosen as vaccines. Wild type nucleotide sequences of the gp160 envelope were codon aligned, manually annotated, and used to reconstruct the phylogeny rooted using a Clade A sequence. Variants from different time points are color-coded as day 189: purple; day 560: red; day 662: blue; day 1729: green. (B) Genetic diversity between clones within each time point was calculated as number of nucleotide substitutions per site between each pair of sequences (i.e. pairwise distance) (C) Genetic divergence based on pairwise distance between clones from each time point and the theoretical Most Recent Common Ancestor illustrates the evolution of subject QA255’s quasispecies over time.
Figure 2
Figure 2. QA255 quasispecies variants evolving under diversifying selection
(A) Codon-specific analysis of positive selection. The red * indicates a posterior probability greater than 95% of dN/dS >1 (dN/dS described as Ka/Ks). Variable regions are highlighted in yellow. Boundary between gp120 and gp41 regions of gp160 is outlined beneath graph. (B) Phylogeny showing the distribution of estimated dN/dS per lineage. Four categories of dN/dS values (10000, 2.212, 0.633, and 0.219) were approximated according to the best-fitting model. Lineages are color-coded based on their assignment to each of these dN/dS categories and the percentage represents the posterior probability that dN/dS > 1.
Figure 3
Figure 3. QA255 Env vaccine sequences, immunization approaches, and Env-specific binding antibodies
(A) Highlighter plot illustrates accumulating non-synonymous changes in the sequence occurring longitudinally in the WT quasispecies variants incorporated in the various immunizations. Nucleotide differences from the Most Recent Common Ancestor sequence are indicated by tick marks (green, silent; red, non-silent; grey, gap). Codon location of the env gene is shown on the X-axis. Brackets to the right outline clones used for immunization on respective weeks. (B) Immunization Strategies. Rabbits were vaccinated with four either MO or WT DNA primes at weeks (brown arrows) 0, 4, 12, and 20, followed by two combination MO 1729O DNA plus LCONS protein boosts in the presence of PEI adjuvant (green arrows) on weeks 29 and 35. (C) LCONS Envelope-specific binding antibodies elicited by the immunization strategies. Relative antibody concentrations of serum samples collected two weeks after each immunization were measured by kinetic ELISA against trimeric LCONS gp140. MO Dual-clone Sequential (MO Dual-clone), black; MO Multi-clone Sequential (MO Multi-clone), blue; WT Multi-clone Sequential (WT Multi-clone), red. Dotted line represents 2.5× pre-immune titers.
Figure 4
Figure 4. Heterologous rabbit humoral response against sensitive Clades A and B viruses
Serial dilutions of sera from week 31 (post first DNA plus protein boost) was tested for neutralization against sensitive Clades A and B heterologous clones HIV-1Q461d1 (A-C) and HIV-1SF162 (D-F). Individual rabbit samples are shown with different symbols for inter-virus comparison. Thick broken line indicates pool pre-immune serum neutralization values. Thin dotted line demarks 50% neutralization. (G & H) Mean ± SEM neutralization values obtained at single dilution (1:40) of rabbit sera was plotted longitudinally. Columns: MO Dual-clone Sequential, black/left; MO Multi-clone Sequential, blue/middle; WT Multi-clone Sequential, red/right. Black arrows represent DNA immunizations, gray arrows represent DNA plus protein boosts.blue/middle; WT Multi-clone Sequential, red/right. Black arrows represent DNA immunizations, gray arrows represent DNA plus protein boosts.
Figure 5
Figure 5. Qualitative assessment of rabbit humoral responses
(A) Avidity ELISA. Avidity indices against LCONS gp140 in samples collected after the final DNA prime for MO groups (week 22) and after the first DNA plus protein boost for WT Multi-clone Sequential (week 31) and after the final DNA plus protein boost (week 37). (B) Comparison of avidity indices between week 37 and week 22 (MO-primed group) and between week 37 and week 31 (WT-primed group) in samples as in top to determine affinity maturation. Asterisks indicate P<0.05 using tests described in the Methods.
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