Immune suppression of challenged vaccinates as a rigorous assessment of sterile protection by lentiviral vaccines

Abstract

We previously reported that an experimental live-attenuated equine infectious anemia virus (EIAV) vaccine, containing a mutated S2 accessory gene, provided protection from disease and detectable infection after virulent virus (EIAV(PV)) challenge [Li F, Craigo JK, Howe L, Steckbeck JD, Cook S, Issel C, et al. A live-attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses. J Virol 2003;77(13):7244-53; Craigo JK, Li F, Steckbeck JD, Durkin S, Howe L, Cook SJ, et al. Discerning an effective balance between equine infectious anemia virus attenuation and vaccine efficacy. J Virol 2005;79(5):2666-77]. To determine if attenuated EIAV vaccines actually prevent persistent infection by challenge virus, we employed a 14-day dexamethasone treatment of vaccinated horses post-challenge to suppress host immunity and amplify replication levels of any infecting EIAV. At 2 months post-challenge the horses were all protected from virulent-virus challenge, evidenced by a lack of EIA signs and detectable challenge plasma viral RNA. Upon immune suppression, 6/12 horses displayed clinical EIA. Post-immune suppression characterizations demonstrated that the attenuated vaccine evidently prevented detectable challenge virus infection in 50% of horses. These data highlight the utility of post-challenge immune suppression for evaluating persistent viral vaccine protective efficacy.

Figure 1. Details of the construction of the nucleotide-deleted live-attenuated vaccine candidate

Schematic representation of EIAV genome, S2 gene, and mutant clones derived from EIAV_UKΔS2. The genomic structure of EIAV provirus is shown at the top. The complete deduced amino acid sequence of the putative S2 protein is shown in single-letter amino acid code at the bottom. The residues in which the original stop codons were introduced into various positions in the EIAV S2 gene are shadowed and underlined. The region where the true nucleotide deletions were generated are indicated below the S2 amino acid sequence. The EIAV_UKΔS2 sequence is displayed at the top and the deletion clones are aligned with it. Specific genomic regions considered during development of deletion mutants are indicated above the EIAV_UKΔS2 sequence (Materials and Methods). The G⁵ stop codon in the SpeI diagnostic site (shaded) is boxed.

Figure 2. Clinical and virological profiles of EIAV_D9 vaccinated horses

Twelve horses (Panels A-D) were inoculated with either 10³ TCID₅₀ EIAV_D9 I.V., with 103 TCID₅₀ EIAV_D9 I.M. (Panels E-H), or with 10⁵ TCID₅₀ EIAV_D9 I.M. (Panels I-L) as described in Materials and Methods (⬇Vax⬇). Rectal temperature ( , 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 using the LDME protocol (DOC,↓↓↓). The period of dexamethasone-induced immune suppression is demarcated by a pink shaded box. Two naïve control animals (Panels M-N) were also challenged with the LDME protocol (DOC,↓↓↓). Febrile episodes were defined by a rectal temperature above 39°C ( ) in conjunction with thrombocytopenia (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).

Figure 3. Delayed-type hypersensitivity analysis of EIAV_D9 vaccinated, immune suppressed animals

Immune status of dexamethasone treated animals was monitored through a delayed-type hypersensitivity assay. DTH responses were determined by subcutaneous injection of 50μg of PHA in 1ml of saline and 1ml of saline alone into the necks of the horses. Twenty-four hours post-injection the diameter of the reaction was measured using constant tension calipers. The control reaction (saline alone) was divided into the PHA reaction to yield the DTH ratio (Y axis). PIS, Pre-Immune suppression; IS, Immune suppression; DPV, days post vaccination.

Figure 4. Development of envelope-specific antibody responses to the EIAV_D9 vaccine

Longitudinal characterization of the quantitative and qualitative properties of induced EIAV envelope-specific antibodies were conducted in ConA ELISA assays of (A) endpoint titer, (B) avidity, and (C) conformational dependence as described in Materials and Methods. (A) Mean serum antibody titers for each time point are presented as the log₁₀ of the highest reciprocal dilution yielding reactivity two standard deviations above background. (B) Mean avidity index measurements are presented as percentages of the antibody-antigen complexes resistant to disruption with 8M urea. (C) Mean conformation dependence values are calculated as the ratio of serum antibody reactivity with native envelope compared to denatured envelope antigen. Conformation ratios greater than 1.0 indicate predominant antibody specificity for conformational determinants, while ratios less than 1.0 indicate predominant antibody specificity for linear envelope determinants. PI, protected from infection (■); UPI, unprotected from infection ( ).

Figure 5. Characterization of virus-specific serum neutralization and cytolytic T-cell activity in horses vaccinated with EIAV_D9

(A) 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 and four weeks post challenge, as described in Materials and Methods. The line (–) denotes the cut off (≥15) value for valid 50% neutralization titers. (B) EIAV Env- and Gag-specific CTL activity elicited by experimental immunization and challenge was measured using fresh PBMC from experimental horses at the day of challenge. The EIAV Env- and Gag-specific CTL activity is presented as percent specific lysis of target cells. The line (–) denotes the cut off (≥10) value for valid specific lysis. *, designates animals protected from disease upon chemical immune suppression.