Envelope determinants of equine lentiviral vaccine protection

Lentiviral envelope (Env) antigenic variation and associated immune evasion present major obstacles to vaccine development. The concept that Env is a critical determinant for vaccine efficacy is well accepted, however defined correlates of protection associated with Env variation have yet to be determined. We reported an attenuated equine infectious anemia virus (EIAV) vaccine study that directly examined the effect of lentiviral Env sequence variation on vaccine efficacy. The study identified a significant, inverse, linear correlation between vaccine efficacy and increasing divergence of the challenge virus Env gp90 protein compared to the vaccine virus gp90. The report demonstrated approximately 100% protection of immunized ponies from disease after challenge by virus with a homologous gp90 (EV0), and roughly 40% protection against challenge by virus (EV13) with a gp90 13% divergent from the vaccine strain. In the current study we examine whether the protection observed when challenging with the EV0 strain could be conferred to animals via chimeric challenge viruses between the EV0 and EV13 strains, allowing for mapping of protection to specific Env sequences. Viruses containing the EV13 proviral backbone and selected domains of the EV0 gp90 were constructed and in vitro and in vivo infectivity examined. Vaccine efficacy studies indicated that homology between the vaccine strain gp90 and the N-terminus of the challenge strain gp90 was capable of inducing immunity that resulted in significantly lower levels of post-challenge virus and significantly delayed the onset of disease. However, a homologous N-terminal region alone inserted in the EV13 backbone could not impart the 100% protection observed with the EV0 strain. Data presented here denote the complicated and potentially contradictory relationship between in vitro virulence and in vivo pathogenicity. The study highlights the importance of structural conformation for immunogens and emphasizes the need for antibody binding, not neutralizing, assays that correlate with vaccine protection

Envelope Determinants of Equine Lentiviral Vaccine Protection

Lentiviral envelope (Env) antigenic variation and associated immune evasion present major obstacles to vaccine development. The concept that Env is a critical determinant for vaccine efficacy is well accepted, however defined correlates of protection associated with Env variation have yet to be determined. We reported an attenuated equine infectious anemia virus (EIAV) vaccine study that directly examined the effect of lentiviral Env sequence variation on vaccine efficacy. The study identified a significant, inverse, linear correlation between vaccine efficacy and increasing divergence of the challenge virus Env gp90 protein compared to the vaccine virus gp90. The report demonstrated approximately 100% protection of immunized ponies from disease after challenge by virus with a homologous gp90 (EV0), and roughly 40% protection against challenge by virus (EV13) with a gp90 13% divergent from the vaccine strain. In the current study we examine whether the protection observed when challenging with the EV0 strain could be conferred to animals via chimeric challenge viruses between the EV0 and EV13 strains, allowing for mapping of protection to specific Env sequences. Viruses containing the EV13 proviral backbone and selected domains of the EV0 gp90 were constructed and in vitro and in vivo infectivity examined. Vaccine efficacy studies indicated that homology between the vaccine strain gp90 and the N-terminus of the challenge strain gp90 was capable of inducing immunity that resulted in significantly lower levels of post-challenge virus and significantly delayed the onset of disease. However, a homologous N-terminal region alone inserted in the EV13 backbone could not impart the 100% protection observed with the EV0 strain. Data presented here denote the complicated and potentially contradictory relationship between in vitro virulence and in vivo pathogenicity. The study highlights the importance of structural conformation for immunogens and emphasizes the need for antibody binding, not neutralizing, assays that correlate with vaccine protection. © 2013 Craigo et al

Phylogenetic analyses of the ConEnv, all consensus Envs, and the longitudinal isolates derived from experimental infection utilized in consensus gene development.

<p>A phylogenetic tree of aligned plasma amino acid sequences (gp90 regions) was constructed by the neighbor joining method from Kimura corrected distances with the optimality criterion set to distance. The tree was rooted to the ancestral Env, EV0. Bootstrap values were determined over 1000 iterations and are indicated at the nodes of the branches. Branch lengths are proportional to the distance existing between the sequences.</p

Viral loads in vaccinated ponies and naïve ponies present distinguishing differences.

<p>The longitudinal average of the viral loads, quantitated from plasma RNA, for the challenged vaccinates as well as the challenged unvaccinated ponies were plotted as a function of the observation period (approximately 300 days). Red symbols in both groups indicate a single animal’s viral level during febrile episode. DOC, Day of challenge; *, Mann-Whitney statistical results, <i>P</i> = 0.015.</p

EVN_Term chimeric viral strain clinical disease is significantly different than disease in unvaccinated controls.

<p>The percentage of EIAV_D9 vaccinated and unvaccinated control animals that were protected from clinical EIA upon challenge with EV_NTerm were plotted as a function of days post-infection. Kaplan-Meier survival plots were generated plotting protection from disease as “0” at the end of the study (120 days post challenge) and disease as “1” on the first day of disease in GraphPad Prism 6.0a (GraphPad Software Inc., LaJolla, Ca.). Gehan-Breslow-Wilcoxon test for statistical relevance demonstrated a significant difference in the survival curves, <i>P</i> = 0.0054.</p

Comparison of deduced amino acid gp90 variable region sequences from EIAV variant Envs (used in attenuated and challenge strain construction) and ConEnv.

<p>The region of <i>env</i> gene coding for the surface glycoprotein, gp90, of the Env variants, EV0, EV6, and EV13 (ref) was compared to the ConEnv sequence. Deduced amino acid sequences were aligned and compared to EV0 (ancestral Env that is analogous to the EIAV_D9 Env). Only the amino acid residues different from EV0 are reported on the alignment. Dots (·) indicate residues identical to the EV0 sequence; dashes (-) indicate amino acid deletion; Previously described variable regions V1 through V8 are boxed and shaded in blue.</p

Characterization of vaccine immune responses prior to challenge.^*

<p>*Numbers represent averages for each trial group, All values in () are SD.</p>a<p>Serum Neutralization titer analyses were against EIAV_PV. Positive neutralization determined as reciprocal titers >25 (27, 40).</p>b<p>Pooled responses of all vaccinate-specific peptides; RQ, Relative Quantity, determined as outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004610#s4" target="_blank">Materials and Methods</a>.</p><p>INFγ, Interferon-γ; GrzB, Granzyme B.</p><p>Characterization of vaccine immune responses prior to challenge.^{<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004610#nt104" target="_blank">*</a>}</p

Clinical and virological profiles of EVN_Term challenged vaccinated and naïve ponies.

<p>The profiles depicted in A–I display the clinical and virological outcomes observed in EIAV_D9 vaccinated animals (<b>A–E</b>), and EIAV naïve animals (<b>F–I</b>) upon challenge with 10³ TCID₅₀ EV_NTerm chimeric proviral strain. (<b>A–E</b>) Five EIAV-naïve ponies were vaccinated with 10³ TCID₅₀ EIAV_D9 I.V. (↑ Vax ↑). Rectal temperature (<b>–</b>, right Y axis) and platelet counts (–-, first left Y axis) were followed daily for up to 300 days (X-axis) after the first vaccine dose. Quantification of the virus load (⧫, second left Y axis) was performed on viral RNA extracted from plasma at periodic time points prior to and after virulent virus challenge seven months post-first vaccination with 10³ TCID₅₀ EV_NTerm, I.V. (↑Challenge). (<b>F–I</b>) Four EIAV-naïve ponies were also challenged with 10³ TCID₅₀ EV_NTerm, I.V. (↑Challenge). Febrile episodes were defined by a achieving a combination of two-three features such as: rectal temperature above 39°C in conjunction with thrombocytopenia (platelet decrease of ≥70,000/µl of whole blood), EIAV viral load ≥10⁵ as well as other clinical signs of EIA.</p

Chimeric strain infections did not result consistently in clinical disease.

<p>The percentage of animals within each trial group, EV_NTerm and EV_CTerm that did not develop clinical EIA was plotted as a function of days post-infection.</p

Neutralization profiles of proviral variant strains with serum from animals experimentally infected with EV0, EV6, EV13, or EVCon.

<p>Variant proviral strains were tested for <i>in vivo</i> pathogenecity through experimental infections of ponies. Serum from the infections was utilized here to test for immunogenicity and characterize the variant Envs. At six-months post-infection serum from two representative animals (labeled A,B) was tested in our standard neutralization assay. The mean reciprocal dilutions of serum which neutralized 50% of input EV0, EV6, EV13, or EVCon as measured in an infectious center assay are presented. The dashed line denotes the cut off (≥25) value for valid 50% neutralization titers.</p