Subunit rotavirus vaccine administered parenterally to rabbits induces active protective immunity

Abstract

Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. The immunogenicity and protective efficacy of different formulations of VLPs administered parenterally to rabbits were tested. Two doses of VLPs (2/6-, G3 2/6/7-, or P[2], G3 2/4/6/7-VLPs) or SA11 simian rotavirus in Freund's adjuvants, QS-21 (saponin adjuvant), or aluminum phosphate (AlP) were administered. Serological and mucosal immune responses were evaluated in all vaccinated and control rabbits before and after oral challenge with 10(3) 50% infective doses of live P[14], G3 ALA lapine rotavirus. All VLP- and SA11-vaccinated rabbits developed high levels of rotavirus-specific serum and intestinal immunoglobulin G (IgG) antibodies but not intestinal IgA antibodies. SA11 and 2/4/6/7-VLPs afforded similar but much higher mean levels of protection than 2/6/7- or 2/6-VLPs in QS-21. The presence of neutralizing antibodies to VP4 correlated (P < 0.001, r = 0.55; Pearson's correlation coefficient) with enhanced protection rates, suggesting that these antibodies are important for protection. Although the inclusion of VP4 resulted in higher mean protection levels, high levels of protection (87 to 100%) from infection were observed in individual rabbits immunized with 2/6/7- or 2/6-VLPs in Freund's adjuvants. Therefore, neither VP7 nor VP4 was absolutely required to achieve protection from infection in the rabbit model when Freund's adjuvant was used. Our results show that VLPs are immunogenic when administered parenterally to rabbits and that Freund's adjuvant is a better adjuvant than QS-21. The use of the rabbit model may help further our understanding of the critical rotavirus proteins needed to induce active protection. VLPs are a promising candidate for a parenterally administered subunit rotavirus vaccine.

FIG. 1

Serum and intestinal antibody responses following two parenteral immunizations (0 and 21 dpv) of rabbits with formalin-inactivated SA11 rotavirus, 10 or 20 μg of G3 2/4/6/7-VLPs, or MA104 cell lysate in either Freund’s adjuvants or AlP. Total (IgM, IgG, and IgA) serum (A), intestinal IgA (B), and intestinal IgG (C) antirotavirus antibodies were measured by ELISA before (56 dpv) and after (84 dpv) oral challenge with lapine ALA rotavirus. Formulation of vaccine and adjuvant are indicated along the x axis. Antibody titers were measured for individual rabbits, and results are plotted as the GMTs of the groups. Error bars represent 1 standard error of the mean. For titers of <50 and <5, 25 and 2.5, respectively, were used to calculate the GMT. A GMT of 25 or 2.5, for serum or intestinal antibodies, respectively, was considered negative. A significant (P < 0.05, Wilcoxon signed ranks test) increase in rotavirus antibody GMTs following challenge is indicated by an asterisk.

FIG. 2

Protection from lapine ALA rotavirus challenge in rabbits immunized twice parenterally with formalin-inactivated SA11 rotavirus, 10 or 20 μg of G3 2/4/6/7-VLPs, or MA104 cell lysate in either Freund’s adjuvant or AlP. Virus antigen shedding curves were plotted for individual rabbits, and the mean area under the curve was calculated for each group. Results are plotted as percent reduction in shedding for each individual animal (•), as well as the mean for each vaccine group (—), calculated by comparing the mean area under the curve for each vaccine group to the mean area under the curve for the control group. The number of rabbits with overlapping levels of protective efficacy is indicated next to the dots (n = 2). Formulation of vaccine, adjuvant, and number of rabbits per group are indicated along the x axis.

FIG. 3

Serum and intestinal antibody responses following two parenteral immunizations (0 and 21 or 28 dpv) of rabbits with formalin-inactivated SA11 rotavirus, MA104 cell lysate, or 50 μg of G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in Freund’s adjuvants (A to C) and with 50 μg of live/psoralen-inactivated SA11 rotavirus, TNC buffer, or 50 μg of G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in QS-21 (D to F). Total (IgM, IgG, and IgA) serum (A and D), intestinal IgG (B and E), and intestinal IgA (C and F) antirotavirus antibodies were measured by ELISA before (56 or 69 dpv) and after (84 or 97 dpv) oral challenge with lapine ALA rotavirus. Antibody titers were measured for individual rabbits, and results are plotted as GMTs of the groups. The GMT of intestinal IgG antibody titers for both 0 and 28 dpc of the rabbits vaccinated with 2/6-VLPs in Freund’s adjuvants is 368. Therefore, for this group, only one box appears on the graph (B). Error bars represent 1 standard error of the mean. For titers of <50 and <5, 25 and 2.5, respectively, were used to calculate the GMT. A GMT of 25 or 2.5, for serum or intestinal antibodies, respectively, was considered negative. A significant (P < 0.05, Wilcoxon signed ranks test) increase in rotavirus antibody GMTs following challenge is indicated by an asterisk.

FIG. 4

Protection from lapine ALA rotavirus challenge in rabbits immunized twice parenterally with formalin-inactivated SA11 rotavirus, MA104 cell lysate, or 50 μg of G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in Freund’s adjuvant (A) or with 50 μg of live/psoralen-inactivated SA11 rotavirus, TNC buffer, or 50 μg of G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in QS-21 (B). Virus antigen shedding curves were plotted for individual rabbits, and the mean area under the curve calculated for each group. Results are plotted as percent reduction in shedding for each individual animal (•), as well as the mean for each vaccine group (—), calculated by comparing the mean area under the curve for each vaccine group to the mean area under the curve for the control group. The number of rabbits with overlapping levels of protective efficacy is indicated next to the dots (n = 2 or 3); formulation of vaccine and number of rabbits per group are indicated along the x axis.

FIG. 5

Serum immune responses to individual rotavirus proteins before (56 or 69 dpv) or after (84 or 97 dpv) oral challenge with lapine ALA rotavirus. Representative results are shown for rabbits vaccinated parenterally either with 2/4/6/7-VLPs in Freund’s adjuvants (F) or with 2/4/6/7-, 2/6/7-, or 2/6-VLPs, psoralen-inactivated SA11, or TNC buffer in QS-21 (Q). Proteins in a SA11-infected cell lysate were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with MAbs directed to VP4, VP6, or VP7, with a hyperimmune serum to SA11, or with paired sera from rabbits immunized with the indicated vaccine formulations. Percent protection from infection for individual rabbits is indicated below each lane. Locations of the viral proteins are indicated on the left.

FIG. 6

Correlation of higher protection rates with presence of serum N-Abs to VP4. (A) Serum N-Ab responses following two parenteral immunizations (0 and 28 dpv) of rabbits with 50 μg of P[2], G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in Freund’s adjuvants or with 50 μg of live or psoralen-inactivated SA11, TNC buffer, or 50 μg of P[2], G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in QS-21 were measured by FFNA against rotavirus strains SA11 (P[2], G3) and B223 (P8[11], G10) and against reassortant virus strains R-N33 (P8[11], G3) and R-A32 (P[2], G10) prior to (56 or 69 dpv) oral challenge with lapine ALA rotavirus. Serum N-Ab titers were measured for individual rabbits, and results are plotted as the GMTs of the groups. Error bars represent 1 standard error of the mean. For titers of <100, 50 was used to calculate the GMT. A GMT of 50 and its log were considered negative. A significant difference (P ≤ 0.021, Mann-Whitney U test) in VP4-specific N-Abs induced by 2/4/6/7-VLPs vaccines measured by comparing serum N-Abs measured against B223 and R-A32 is indicated by an asterisk. (B) Scatterplot of the log of the SA11 VP4-specific-NAb titers and the percent reduction of virus antigen shedding for individual rabbits (n = 58). Results were graphed to indicate whether rabbits were immunized with both VP4 and VP7 (■; SA11 or 2/4/6/7-VLPs in either adjuvant), absence of VP4 but presence of VP7 (•; 2/6/7-VLPs in either adjuvant), or absence of both VP4 and VP7 (▴; 2/6-VLPs in either adjuvant or TNC buffer in QS-21). Correlation coefficient was calculated by Pearson’s correlation coefficient.

FIG. 7

Correlation between reduction of virus antigen shedding as measured by ELISA and reduction of infectious virus titers as measured by FFA independently of vaccine formulation administered to rabbits. (A) Scatterplot of the areas under the curve for virus antigen shedding (as measured by ELISA) versus the log of infectious virus titers (as measured by FFA). Analysis was performed on a subset of rabbits (n = 17) vaccinated parenterally with buffer, SA11 virus, or different VLP formulations. Correlation coefficient was calculated by Pearson’s correlation coefficient. Line depicts linear regression analysis [f(x) = 0.247X − 0.969]. Boxes indicate clusters of rabbits according to percent protection ranges. (B) Daily mean infectious (0 to 10 dpc) ALA rotavirus titers measured by FFA were determined for rabbits grouped based on protection, independent of vaccine formulation administered to rabbits. Based on the scatterplot, rabbits were grouped based on 0% (■), 20 to 65% (•), 68 to 98% (▴), and 100% (⧫) protection as measured by ELISA. For infectious titers of <10, 5 was used to calculate the mean infectious virus titer. An infectious titer of 5 was considered negative.