Discerning an effective balance between equine infectious anemia virus attenuation and vaccine efficacy

Abstract

Among the diverse experimental vaccines evaluated in various animal lentivirus models, live attenuated vaccines have proven to be the most effective, thus providing an important model for examining critical immune correlates of protective vaccine immunity. We previously reported that an experimental live attenuated vaccine for equine infectious anemia virus (EIAV), based on mutation of the viral S2 accessory gene, elicited protection from detectable infection by virulent virus challenge (F. Li et al., J. Virol. 77:7244-7253, 2003). To better understand the critical components of EIAV vaccine efficacy, we examine here the relationship between the extent of virus attenuation, the maturation of host immune responses, and vaccine efficacy in a comparative study of three related attenuated EIAV proviral vaccine strains: the previously described EIAV(UK)DeltaS2 derived from a virulent proviral clone, EIAV(UK)DeltaS2/DU containing a second gene mutation in the virulent proviral clone, and EIAV(PR)DeltaS2 derived from a reference avirulent proviral clone. Inoculations of parallel groups of eight horses resulted in relatively low levels of viral replication (average of 10(2) to 10(3) RNA copies/ml) and a similar maturation of EIAV envelope-specific antibody responses as determined in quantitative and qualitative serological assays. However, experimental challenge of the experimentally immunized horses by our standard virulent EIAV(PV) strain by using a low-dose multiple exposure protocol (three inoculations with 10 median horse infective doses, administered intravenously) revealed a marked difference in the protective efficacy of the various attenuated proviral vaccine strains that was evidently associated with the extent of vaccine virus attenuation, time of viral challenge, and the apparent maturation of virus-specific immunity.

FIG. 1.

Clinical and virologic profiles of EIAV_UKΔS2 experimental horses. Eight horses (A to H, respectively) were inoculated with EIAV_UKΔS2 as described in Materials and Methods. Rectal temperature (solid line, right y axis) and platelet counts (dotted line, first left y axis) were monitored 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 by using the LDME protocol (arrows). S2 diagnostic results for each animal are indicated in each respective panel above the date of analysis by either a V (vaccine strain) or a C (challenge strain).

FIG. 2.

Clinical and virologic profiles of EIAV_UKΔS2/DU experimental horses. Eight horses (A to H, respectively) were inoculated with EIAV_UKΔS2/DU as described in Materials and Methods. Rectal temperature (solid line, right y axis) and platelet counts (dotted line, first left y axis) were monitored 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 by using the LDME protocol (arrows). Febrile episodes (see panel A) were defined by a rectal temperature of >39°C in conjunction with a reduction in platelets (< 100,000/μl of whole blood) and other clinical symptoms of EIA. S2 diagnostic results for each animal are indicated in each respective panel above the date of analysis by either a V (vaccine strain) or a C (challenge strain).

FIG. 3.

Clinical and virological profiles of EIAV_PRΔS2 experimental horses. Eight horses (A to H, respectively) were inoculated with EIAV_PRΔS2 as described in Materials and Methods. Rectal temperature (solid line, right y axis) and platelet counts (dotted line, first left y axis) were monitored 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 by using the LDME protocol (arrows). Febrile episodes (E and H) were defined by a rectal temperature of >39°C in conjunction with a reduction in platelets (<100,000/μl of whole blood) and other clinical symptoms of EIA. S2 diagnostic results for each animal are indicated in each respective panel above the date of analysis by either a V (vaccine strain) or a C (challenge strain). *, EHV-associated fevers.

FIG. 4.

Clinical and virologic profiles of control naive horses. Four horses (A to D, respectively) were subjected to challenge with virulent EIAV_PV by using the LDME protocol (see Materials and Methods). Rectal temperature (solid line, right y axis) and platelet counts (dotted line, first left y axis) were monitored daily for up to 300 days (x axis) postchallenge. 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 by using the LDME protocol (arrows). Febrile episodes were defined by a rectal temperature of >39°C in conjunction with a reduction in platelets (<100,000/μl of whole blood) and other clinical symptoms of EIA.

FIG. 5.

Development of envelope-specific antibody responses to various attenuated EIAV vaccines. Longitudinal characterization of the quantitative and qualitative properties of induced EIAV envelope-specific antibodies were conducted in concanavalin A ELISAs of endpoint titer (panels 1), avidity (panels 2), and conformational dependence (panels 3) as described in Materials and Methods. Analyses were performed on allthree trial groups: EIAV_UKΔS2 (A), EIAV_UKΔS2/DU (B), and EIAV_PRΔS2 (C). (D) Average levels for each assessment were also calculated to extricate and simplify comparative analysis. (A.1 to D.1) Antibody titers in serum for each time point are presented as the log₁₀ of the highest reciprocal dilution yielding reactivity two standard deviations above background. (A.2 to D.2) Avidity index measurements are presented as percentages of the antibody-antigen complexes resistant to disruption with 8 M urea. (A.3 to D.3) Conformation dependence values are calculated as the ratio of serum antibody reactivity with native envelope compared to denatured envelope antigen. Conformation ratios of >1.0 indicate predominant antibody specificity for conformational determinants, whereas ratios of <1.0 indicate predominant antibody specificity for linear envelope determinants.

FIG. 6.

Characterization of virus-specific serum neutralization in horses inoculated with EIAV_UKΔS2, EIAV_UKΔS2/DU, or EIAV_PRΔS2. The mean reciprocal dilutions of serum from vaccinated horses which neutralized 50% of input EIAV_PV as measured in an infectious center assay are presented for serum samples collected at the day of challenge (DOC) and 4 weeks postchallenge (PC), as described in Materials and Methods. The line denotes the cutoff (≥10) value for valid 50% neutralization titers.

FIG. 7.

Clinical and virologic profiles of EIAV_UKΔS2 vaccinated horses and naive control horses challenged at 2 months. Five horses (A to E, respectively) were inoculated with EIAV_UKΔS2 and challenged at 3 months after the first vaccine dose, as described in Materials and Methods. Two naive control horses (F and G) were subjected to challenge in parallel with virulent EIAV_{PV by using} the LDME protocol (see Materials and Methods). Rectal temperature (solid line, right y axis) and platelet counts (dotted line, first left y axis) were monitored daily for up to 170 days (x axis) after vaccination. Quantification of the virus load (♦, second left y axis, ⋄, undetectable) was performed on viral RNA extracted from plasma at periodic time points prior to and after virulent virus challenge by using the LDME protocol (arrows). S2 diagnostic results for each animal are indicated in each respective panel above the date of analysis by either a V (vaccine strain) or a C (challenge strain).