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

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.Fil: Vega, Celina Guadalupe. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bok, Marina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vlasova, Anastasia N.. Ohio State University; Estados UnidosFil: Chattha, Kuldeep S.. Ohio State University; Estados UnidosFil: Gómez Sebastián, Silvia. Universidad Politécnica de Madrid; EspañaFil: Nuñez, Carmen. Universidad Politécnica de Madrid; EspañaFil: Alvarado, Carmen. Universidad Politécnica de Madrid; EspañaFil: Lasa, Rodrigo. Universidad Politécnica de Madrid; EspañaFil: Escribano, José M.. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria. Departamento Mejora Genética y Biotecnología; EspañaFil: Garaicoechea, Lorena Laura. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández, Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Bok, Karin. National Institutes of Health; Estados UnidosFil: Wigdorovitz, Andrés. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Saif, Linda J.. Ohio State University; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Vitamin A Deficiency Impairs Adaptive B and T Cell Responses to a Prototype Monovalent Attenuated Human Rotavirus Vaccine and Virulent Human Rotavirus Challenge in a Gnotobiotic Piglet Model

<div><p>Rotaviruses (RV) are a major cause of gastroenteritis in children. Widespread vitamin A deficiency is associated with reduced efficacy of vaccines and higher incidence of diarrheal infections in children in developing countries. We established a vitamin A deficient (VAD) gnotobiotic piglet model that mimics subclinical vitamin A deficiency in children to study its effects on an oral human rotavirus (HRV) vaccine and virulent HRV challenge. Piglets derived from VAD and vitamin A sufficient (VAS) sows were orally vaccinated with attenuated HRV or mock, with/without supplemental vitamin A and challenged with virulent HRV. Unvaccinated VAD control piglets had significantly lower hepatic vitamin A, higher severity and duration of diarrhea and HRV fecal shedding post-challenge as compared to VAS control pigs. Reduced protection coincided with significantly higher innate (IFNα) cytokine and CD8 T cell frequencies in the blood and intestinal tissues, higher pro-inflammatory (IL12) and 2-3 fold lower anti-inflammatory (IL10) cytokines, in VAD compared to VAS control pigs. Vaccinated VAD pigs had higher diarrhea severity scores compared to vaccinated VAS pigs, which coincided with lower serum IgA HRV antibody titers and significantly lower intestinal IgA antibody secreting cells post-challenge in the former groups suggesting lower anamnestic responses. A trend for higher serum HRV IgG antibodies was observed in VAD vs VAS vaccinated groups post-challenge. The vaccinated VAD (non-vitamin A supplemented) pigs had significantly higher serum IL12 (PID2) and IFNγ (PID6) compared to vaccinated VAS groups suggesting higher Th1 responses in VAD conditions. Furthermore, regulatory T-cell responses were compromised in VAD pigs. Supplemental vitamin A in VAD pigs did not fully restore the dysregulated immune responses to AttHRV vaccine or moderate virulent HRV diarrhea. Our findings suggest that that VAD in children in developing countries may partially contribute to more severe rotavirus infection and lower HRV vaccine efficacy.</p> </div

Vitamin A status affected HRV IgA antibody responses post-virulent HRV challenge.

<p>Geometric mean serum (A) and intestinal (B) HRV IgA antibody titers and mean intestinal HRV IgA antibody secreting cells (C,D) in vitamin A deficient (VAD) and sufficient (VAS) gnotobiotic pigs vaccinated with AttHRV vaccine or placebo with or without vitamin A supplementation at pre- (PID26/PCD0) and post (PID36/PCD10)-HRV challenge time-points. Data shown as mean values ± standard error of the mean. Bars with different lower-case letters for HRV IgA antibody titers (A,B) at each time-point (pre- or post-challenge) differ significantly between groups (Duncans multiple range t test on log₁₀ transformed data, <i>p</i> ≤ 0.05). Significant differences between groups for HRV IgA ASC are indicated by capped lines as determined by non-parametric Kruskal-wallis rank sum test (<i>p</i> ≤ 0.05). The arrow (C) indicates increased duodenal IgA ASC in Vac+VitA group compared to Vac group for both VAD and VAS pigs at PID26/PCD0 (pre-challenge). Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only. Pre-challenge: n=8-13 (serum IgA antibodies), 3-7 (intestinal IgA antibodies and ASC), Post-challenge: n=4-7 (serum and intestinal IgA antibodies and ASC).</p

Vaccinated VAS groups had higher frequencies of B cells in ileum (pre-challenge) and duodenum (post-challenge).

<p>Mean frequency of B lymphocytes (CD21⁺CD3^-) among mononuclear cells in vitamin A deficient (VAD) and sufficient (VAS) pigs vaccinated with AttHRV vaccine or placebo with or without vitamin A supplementation in ileum (A), duodenum (B), blood (C) and spleen (D) at pre- (PID26/PCD0) and post (PID36/PCD10)-virulent HRV challenge. Bars represent mean values and standard error of the mean. Significant differences between groups are indicated by capped lines as determined by non-parametric Kruskal-wallis rank sum test (<i>p</i> ≤ 0.05). The dotted rectangles (A) indicate trend for higher frequencies of B cells in vaccinated VAS groups compared to vaccinated VAS groups at pre-challenge. Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only, Pre = pre-challenge (number), Post = post-challenge (number). Pre-challenge: n= 3-7, Post-challenge: n=4-7.</p

Summary of pilot data for different regimens for vitamin A supplementation.

<p>Geometeric mean titers of serum (A) and intestinal (B) HRV IgA antibody titers and mean intestinal HRV IgA antibody secreting cells (C) in AttHRV vaccinated pigs with supplemented of 50,000 IU for 11 days (Vac+VitA multiple doses, n=2) or 100,000 IU at each vaccine time-point (Vac+VitA, n=2). Data shown as mean values ± standard error of the mean. Arrows in a, b, c and d indicate increase in these parameters for Vac+VitA multiple dose compared to Vac+VitA group. Vac+VitA = 3X AttHRV vaccinated + supplemental vitamin A. No statistical analysis was done because of low numbers of pigs tested per treatment in this pilot study.</p

Vitamin A deficient vaccinated groups had lower HRV IgG antibody responses post-virulent HRV challenge.

<p>Geometric mean serum HRV IgG (A) titers in vitamin A deficient (VAD) and sufficient (VAS) gnotobiotic pigs vaccinated with AttHRV vaccine or placebo with or without vitamin A supplementation at pre- (PID26/PCD0) and post (PID36/PCD10)-HRV challenge time-points. Data shown as mean values ± standard error of the mean. Bars with different lower-case letters for HRV IgG antibody titers (A) at each time-point (pre- or post-challenge) differ significantly between groups (Duncans multiple range t test on log₁₀ transformed data, <i>p</i> ≤ 0.05). (B) shows the HRV IgG1:IgG2 ratios for different treatment groups. Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only. Pre-challenge: n=8-13, Post-challenge: n=4-7.</p

Summary of innate and Th17 cytokine responses.

<p>Mean concentrations (± SEM) of IFNα (A,D; innate), IL8 (B,E; innate) and IL17 (C,F; Th17) cytokines and chemokines in serum of vitamin A deficient (VAD) and sufficient (VAS) pigs vaccinated with AttHRV vaccine or placebo with or without vitamin A supplementation. Data shown as mean values ± standard error of the mean. Different lower-case letters indicate statistically significant difference (non-parametric Kruskal-wallis rank sum test<i>, p</i> ≤ 0.05) among the treatment groups belonging to VAD and VAS pigs at the same time-point for each cytokine. The circle (f) indicates increase in IL17 in vaccinated VAS groups compared to VAS control groups. Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only.</p

Vitamin A deficiency induced pro-inflammatory immune responses.

<p>Mean concentrations (± SEM) of IL12 (A,D; Th1), IFNγ (B,E; Th1) and IL10 (C,F; anti-inflammatory) cytokines in serum of vitamin A deficient (VAD) and sufficient (VAS) pigs vaccinated with AttHRV vaccine or placebo with or without vitamin A supplementation. Data shown as mean values ± standard error of the mean. Different lower-case letters indicate statistically significant difference (non-parametric Kruskal-wallis rank sum test<i>, p</i> ≤ 0.05) among the treatment groups belonging to VAD and VAS pigs at the same time-point for each cytokine. The circles (C,F) indicate 2-3 fold higher IL10 in VAS control groups compared to VAD control groups. Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only.</p

IgY antibodies protect against human Rotavirus induced diarrhea in the neonatal gnotobiotic piglet disease model.

Group A Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The aim of the present work was to evaluate protection against rotavirus (RV) diarrhea conferred by the prophylactic administration of specific IgY antibodies (Ab) to gnotobiotic piglets experimentally inoculated with virulent Wa G1P[8] human rotavirus (HRV). Chicken egg yolk IgY Ab generated from Wa HRV hyperimmunized hens specifically recognized (ELISA) and neutralized Wa HRV in vitro. Supplementation of the RV Ab free cow milk diet with Wa HRV-specific egg yolk IgY Ab at a final ELISA Ab titer of 4096 (virus neutralization -VN- titer = 256) for 9 days conferred full protection against Wa HRV associated diarrhea and significantly reduced virus shedding. This protection was dose-dependent. The oral administration of semi-purified passive IgY Abs from chickens did not affect the isotype profile of the pig Ab secreting cell (ASC) responses to Wa HRV infection, but it was associated with significantly fewer numbers of HRV-specific IgA ASC in the duodenum. We further analyzed the pigś immune responses to the passive IgY treatment. The oral administration of IgY Abs induced IgG Ab responses to chicken IgY in serum and local IgA and IgG Ab responses to IgY in the intestinal contents of neonatal piglets in a dose dependent manner. To our knowledge, this is the first study to show that IgY Abs administered orally as a milk supplement passively protect neonatal pigs against an enteric viral pathogen (HRV). Piglets are an animal model with a gastrointestinal physiology and an immune system that closely mimic human infants. This strategy can be scaled-up to inexpensively produce large amounts of polyclonal IgY Abs from egg yolks to be applied as a preventive and therapeutic passive Ab treatment to control RV diarrhea