Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea

Abstract

Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

Figure 1. Mean titer of virus shed per day per pig (from CCIF assay) detected daily in rectal swab fluids of the experimental groups of piglets.

All animals were orally inoculated at 72 h of age [0 post inoculation day (0 PID)], and euthanized at 21±3 PID. SIC: small intestinal contents; and LIC: large intestinal contents, were collected after euthanasia. Horizontal bars represent the mean days of diarrhea duration for each group. The arrow at 0 PID indicates the inoculation day. The thin line indicates the duration of the passive treatments.

Figure 2. Geometric mean isotype-specific Ab titers (GMT) to Wa HRV per group per day (from ELISA assay) detected in rectal swab fluids of the experimental groups of piglets.

All animals were orally inoculated at 72 h of age [0 post inoculation day (0 PID)], and euthanized at 21±3 PID. SIC: small intestinal contents; and LIC: large intestinal contents, were collected after euthanasia. The arrow at 0 PID indicates the inoculation day.

Figure 3. Wa HRV-specific porcine Abs in piglet's sera.

The isotype-specific and VN Abs to Wa HRV in serum samples were determined weekly by ELISA and VN assay. The arrow indicates the experimental inoculation with Wa HRV (0 PID). PID: post inoculation days.

Figure 4. Numbers of Wa HRV-specific ASC per 5×10⁵ MNC obtained from systemic lymphoid tissues (blood and spleen) and MLN draining the small and large intestine at 21 PID.

When comparing ASC numbers of the same isotype among treatment groups, different letters indicate a significant difference (Kruskal-Wallis rank sum test, p<0.05). n = number of piglets in each group. The IgM ASC response was not included as no IgM ASC were detected in most of the groups of pigs.

Figure 5. Numbers of Wa HRV-specific ASC per 5×10⁵ MNC obtained from gut-associated lymphoid tissues (Duodenum, Jejunum, Ileum) 21 PID.

When comparing ASC numbers of the same isotype among treatment groups, different letters indicate a significant difference (Kruskal-Wallis rank sum test, p<0.05). n = number of piglets in each group. The IgM ASC response was not included as no IgM ASC were detected in most of the groups of pigs.

Figure 6. Immune responses to the passive treatments were determined in sera and intestinal contents of treated piglets.

Continuous lines represent geometric mean of porcine IgG Ab titers (GMT) to the corresponding treatment administered (VHH, IgY or IgG Abs) in serum samples per group every 7 days. Dashed lines represent ELISA Ab titer of passive Abs to Wa HRV (VHH, IgY or IgG Abs) in rectal swabs every 7 days. The bars at PID 21 represent porcine IgG and IgA Abs to passive treatment in SIC: small intestinal contents and LIC: large intestinal contents collected after euthanasia. All animals were orally inoculated at 72 h of age [0 post inoculation day (0 PID)], and euthanized at PID 21±3. The arrow at PID 0 indicates the inoculation day. Different letters indicate significant differences between groups (p<0.05). In the case of groups CL, IgG and NT the results represent porcine IgG Ab titers to chicken IgY in serum but a similar response was observed against VHH nanoAbs (data not shown).