Evaluation of Cross-Protection of a Lineage 1 West Nile Virus Inactivated Vaccine against Natural Infections from a Virulent Lineage 2 Strain in Horses, under Field Conditions

Abstract

Although experimental data regarding cross-protection of horse West Nile virus (WNV) vaccines against lineage 2 infections exist, the cross-protective efficacy of these vaccines under field conditions has not been demonstrated. This study was conducted to evaluate the capability of an inactivated lineage 1 vaccine (Equip WNV) to protect against natural infections from the Nea Santa-Greece-2010 lineage 2 strain. In total, 185 WNV-seronegative horses in Thessaloniki, Greece, were selected during 2 consecutive years (2011 and 2012); 140 were immunized, and 45 were used as controls. Horses were examined for signs compatible with WNV infection. Neutralizing antibody titers against the Greek strain and the PaAn001/France lineage 1 strain were determined in immunized horses. WNV circulation was detected during both years in the study area. It was estimated that 37% and 27% of the horses were infected during 2011 and 2012, respectively. Three control animals developed clinical signs, and the WNV diagnosis was confirmed. Signs related to WNV infection were not observed in the vaccinated animals. The nonvaccinated animals had a 7.58% ± 1.82% higher chance of exhibiting signs than immunized animals (P < 0.05). Neutralizing antibodies raised against both strains in all immunized horses were detectable 1 month after the initial vaccination course. The cross-protective capacity of the lowest titer (1:40) was evident in 19 animals which were subsequently infected and did not exhibit signs. Neutralizing antibodies were detectable until the annual booster, when strong anamnestic responses were observed (geometrical mean titer ratio [GMTR] for lineage 1 of 30.2; GMTR for lineage 2 of 27.5). The results indicate that Equip WNV is capable of inducing cross-protection against natural infections from a virulent lineage 2 WNV strain in horses.

FIG 1

Timeline of the immunizations and blood serum samplings performed in horses for the evaluation of the cross-protective immunity offered by the inactivated vaccine. The black syringes indicate the double primary vaccinations; the white syringe indicates the annual booster vaccination. Arrows depict the time points of blood serum samplings. Weeks in which these samplings were conducted are displayed above the arrows. The two black arrows marked with asterisks depict samplings performed 1 week prior to the initiation of the primary vaccinations of 2011 and 2012, respectively, in order to detect and select WNV-seronegative horses.

FIG 2

Neutralizing antibody (NAb) geometrical mean titers (GMTs) of 23 immunized horses which were not infected against the Nea Santa-Greece-2010 lineage 2 strain (gray curve, ◆) as well as the PaAn001/France lineage 1 strain (black curve, ▲). SNTs were performed in sera collected from W0 (the time of the first dose of the double primary vaccination in 2011) and until W52 (i.e., 1 month after the annual booster immunization). Paired t test analysis revealed no significant differences in the NAb GMTs against the two antigens at any sampling point (P > 0.28). Error bars encompass the range of the individual NAb titers against each antigen and for every sampling time point. The geometrical mean titer ratio (GMTR) calculated for the annual booster against each strain is also presented.

FIG 3

Alignment of the E protein amino acid sequences from the vaccine lineage 1 strain New York 1999/VM-2 (GenBank accession no. AF260967), the circulating lineage 2 strain Nea Santa-Greece-2010 (GenBank accession no. HQ537483), and the South African lineage 2 strain SA93/01 (GenBank accession no. EF429198) of West Nile virus. Dots indicate amino acid identities. The domains are indicated by bars, as explained in the legend. The investigation of the immunological similarity among the three peptides revealed no differences between the two lineage 2 strains. The comparison of the lineage 1 (vaccine) and the lineage 2 peptide sequences of the E protein revealed 23 amino acid substitutions.