SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice.

Since the beginning of the COVID-19 pandemic, there has been a significant need to develop antivirals and vaccines to combat the disease. In this work, we developed llama-derived nanobodies (Nbs) directed against the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Most of the Nbs with neutralizing properties were directed to RBD and were able to block S-2P/ACE2 interaction. Three neutralizing Nbs recognized the N-terminal domain (NTD) of the S-2P protein. Intranasal administration of Nbs induced protection ranging from 40% to 80% after challenge with the WA1/2020 strain in k18-hACE2 transgenic mice. Interestingly, protection was associated with a significant reduction in virus replication in nasal turbinates and a reduction in virus load in the brain. Employing pseudovirus neutralization assays, we identified Nbs with neutralizing capacity against the Alpha, Beta, Delta, and Omicron variants, including a Nb capable of neutralizing all variants tested. Furthermore, cocktails of different Nbs performed better than individual Nbs at neutralizing two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest the potential of SARS-CoV-2 specific Nbs for intranasal treatment of COVID-19 encephalitis.

A single nanobody neutralizes multiple epochally evolving human noroviruses by modulating capsid plasticity.

Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a "raised" conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection.

A multispecies competitive nanobody-based ELISA for the detection of antibodies against hepatitis E virus.

The hepatitis E virus (HEV) is an emergent zoonotic virus causing viral hepatitis worldwide. Clinically, hepatitis E is not easily distinguished from other types of acute viral hepatitis. There is a need for HEV diagnostic assays to detect and prevent interspecies transmission among susceptible populations. Nanobodies (Nbs) are expressed recombinantly in different systems, produced with high yields, and have superior physicochemical properties compared with conventional antibodies (Ab). Several Nbs against ORF2, the capsid protein and main antigen, were selected and produced in E. coli. Nb39 and Nb74 specifically recognized HEV ORF2 (genotypes 3 and 4). A competitive ELISA (cELISA) was developed and validated using a reference panel of human (n = 86) and swine sera (n = 116) tested in comparison with a commercial kit. The optimal cutoff values determined by ROC analysis were 69.16% (human) and 58.76% (swine); the sensitivity and specificity were high: 97.4% (95% CI 86.5-99.5%) and 95.8% (95% CI 86.0-98.8%) for human vs. 100% (95% CI 93.5-100%) and 98.3% (95% CI 91.0-99.7%) for swine. Further, the cELISA detected total anti-HEV antibodies in wild boar, deer, and mice. To our knowledge, this is the first report of production of Nbs against HEV-3 ORF2 for diagnostic purposes.

Development of Nanobody-Displayed Whole-Cell Biosensors for the Colorimetric Detection of SARS-CoV-2.

The accurate and effective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to preventing the spread of infectious diseases and ensuring human health. Herein, a nanobody-displayed whole-cell biosensor was developed for colorimetric detection of SARS-CoV-2 spike proteins. Serving as bioreceptors, yeast surfaces were genetically engineered to display SARS-CoV-2 binding of llama-derived single-domain antibodies (nanobodies) with high capture efficiency, facilitating the concentration and purification of SARS-CoV-2. Gold nanoparticles (AuNPs) employed as signal transductions were functionalized with horseradish peroxidase (HRP) and anti-SARS monoclonal antibodies to enhance the detection sensitivity. In the presence of SARS-CoV-2 spike proteins, the sandwiched binding will be formed by linking engineered yeast, SARS-CoV-2 spike proteins, and reporter AuNPs. The colorimetric signal was generated by the enzymatic reaction of HRP and its corresponding colorimetric substrate/chromogen system. At the optimal conditions, the developed whole-cell biosensor enables the sensitive detection of SARS-CoV-2 spike proteins in a linear range from 0.01 to 1 µg/mL with a limit of detection (LOD) of 0.037 µg/mL (about 4 × 108 virion particles/mL). Furthermore, the whole-cell biosensor was demonstrated to detect the spike protein of different SARS-CoV-2 variants in human serum, providing new possibilities for the detection of future SARS-CoV-2 variants.

Development of an IgY-Based Treatment to Control Bovine Coronavirus Diarrhea in Dairy Calves.

Bovine Coronavirus (BCoV) is a major pathogen associated with neonatal calf diarrhea. Standard practice dictates that to prevent BCoV diarrhea, dams should be immunized in the last stage of pregnancy to increase BCoV-specific antibody (Ab) titers in serum and colostrum. For the prevention to be effective, calves need to suck maternal colostrum within the first six to twelve hours of life before gut closure to ensure a good level of passive immunity. The high rate of maternal Ab transfer failure resulting from this process posed the need to develop alternative local passive immunity strategies to strengthen the prevention and treatment of BCoV diarrhea. Immunoglobulin Y technology represents a promising tool to address this gap. In this study, 200 laying hens were immunized with BCoV to obtain spray-dried egg powder enriched in specific IgY Abs to BCoV on a large production scale. To ensure batch-to-batch product consistency, a potency assay was statistically validated. With a sample size of 241, the BCoV-specific IgY ELISA showed a sensitivity and specificity of 97.7% and 98.2%, respectively. ELISA IgY Abs to BCoV correlated with virus-neutralizing Ab titers (Pearson correlation, R2 = 0.92, p < 0.001). Most importantly, a pilot efficacy study in newborn calves showed a significant delay and shorter duration of BCoV-associated diarrhea and shedding in IgY-treated colostrum-deprived calves. Calves were treated with milk supplemented with egg powder (final IgY Ab titer to BCoV ELISA = 512; VN = 32) for 14 days as a passive treatment before a challenge with BCoV and were compared to calves fed milk with no supplementation. This is the first study with proof of efficacy of a product based on egg powder manufactured at a scale that successfully prevents BCoV-associated neonatal calf diarrhea.

Nanobodies against SARS-CoV-2 reduced virus load in the brain of challenged mice and neutralized Wuhan, Delta and Omicron Variants.

In this work, we developed llama-derived nanobodies (Nbs) directed to the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Nanobodies were selected after the biopanning of two VHH-libraries, one of which was generated after the immunization of a llama (lama glama) with the bovine coronavirus (BCoV) Mebus, and another with the full-length pre-fused locked S protein (S-2P) and the RBD from the SARS-CoV-2 Wuhan strain (WT). Most of the neutralizing Nbs selected with either RBD or S-2P from SARS-CoV-2 were directed to RBD and were able to block S-2P/ACE2 interaction. Three Nbs recognized the N-terminal domain (NTD) of the S-2P protein as measured by competition with biliverdin, while some non-neutralizing Nbs recognize epitopes in the S2 domain. One Nb from the BCoV immune library was directed to RBD but was non-neutralizing. Intranasal administration of Nbs induced protection ranging from 40% to 80% against COVID-19 death in k18-hACE2 mice challenged with the WT strain. Interestingly, protection was not only associated with a significant reduction of virus replication in nasal turbinates and lungs, but also with a reduction of virus load in the brain. Employing pseudovirus neutralization assays, we were able to identify Nbs with neutralizing capacity against the Alpha, Beta, Delta and Omicron variants. Furthermore, cocktails of different Nbs performed better than individual Nbs to neutralize two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest these Nbs can potentially be used as a cocktail for intranasal treatment to prevent or treat COVID-19 encephalitis, or modified for prophylactic administration to fight this disease.

Assessment of Influential Factors for Scours Associated with Cryptosporidium sp., Rotavirus and Coronavirus in Calves from Argentinean Dairy Farms.

Scours is the most common disease in dairy calves, and it is a multifactorial syndrome complex. Cryptosporidium sp. (C. sp.), rotavirus group A (RVA), and bovine coronavirus (BCoV) are the three main pathogens associated with scours. The objective of this study was to identify potential factors associated with scours, C. sp., RVA, and BCoV infections in preweaned dairy calves from Lerma Valley in Salta Province, Argentina. A total of 488 preweaned calves from 19 dairy farms located in the Lerma Valley were enrolled in this observational study. One fecal sample was collected from each calf between one week and two months of age for assessment of C. sp., RVA, and BCoV infection status. Cryptosporidium sp. oocysts and RVA and BCoV antigens in fecal samples were assessed using microscopic observation and indirect enzyme-linked immune sorbent assay (iELISA), respectively. A voluntary questionnaire was developed and used to collect data regarding management practices from the participants' farms. The data were analyzed using multivariable logistic regression models. Scours incidence was 35.4%, and a greater proportion of calves younger than 20 days were affected. Of the fecal samples, 18% and 9.5% tested were positives for C. sp. and RVA, respectively, while BCoV was detected only in two calves. Furthermore, 84.2% and 63.1% of the farms tested positive for Cryptosporidium sp. and RVA, respectively. In addition, the following variables were associated with higher odds of having scours: (1) herd size (>300 milking cows; OR = 1.7), (2) calf age (<20 days of age; OR = 2.2), (3) RVA and C. sp. test (positive test; RVA OR = 2.6; C. sp. OR = 3), calf feeding practices (feeding milk replacer; OR = 1.81), and newborn calf management practices (calf moved from maternity pen <6 h after calving; OR = 1.7). Concerning RVA infection, calves less than 20 days of age (OR = 2.6) had a higher chance of testing positive for RVA, while calves that remained in the calving pen for less than 6 h after calving had a lower chance (OR = 0.3). On the other hand, for C. sp. infection, large farm size (>300 milking cows; OR = 1.2) and young calf age (<20 days of age; OR = 4.4) indicated a higher chance of testing positive for C. sp., while calves belonging to farms that fed frozen colostrum (OR = 0.2) had a lower chance of becoming infected with C. sp. The result of this study indicated that scours is a prevalent disease in farms of the Lerma Valley, Salta, Argentina, and that RVA and C. sp. infections, along with specific farm management practices, might be important contributing factors that could increase the chance of NCS in dairy farms.

Occurrence of Cryptosporidium and other enteropathogens and their association with diarrhea in dairy calves of Buenos Aires province, Argentina.

Cryptosporidiosis of neonatal dairy calves causes diarrhea, resulting in important economic losses. In Argentina, prevalence values of Cryptosporidium spp. and other enteropathogens such as group A rotavirus (RVA), bovine coronavirus (BCoV) and enterotoxigenic Escherichia coli (ETEC, endotoxin STa+), have been independently studied in different regions. However, an integrative epidemiological investigation on large-scale farms has not been carried out. In this study, fecal samples (n = 908) were randomly collected from diarrheic and healthy calves from 42 dairy farms, and analyzed for the presence of Cryptosporidium spp., RVA, BCoV, ETEC (STa+) and Salmonella spp. In all sampled dairy farms, dams had been vaccinated against rotavirus and gram-negative bacteria to protect calves against neonatal diarrhea. The proportion of calves shedding Cryptosporidium spp., RVA, and BCoV in animals younger than 20 days of age were 29.8%, 12.4% and 6.4%, and in calves aged between 21 and 90 days, 5.6%, 3.9%, and 1.8%, respectively. ETEC was absent in the younger, and occurred only sporadically in the older group (0.9%), whereas Salmonella spp. was absent in both. The observed sporadic finding or even absence of bacterial pathogens might be explained by the frequent use of parenteral antibiotics in 25.3% and 6.5% of the younger and the older group of calves, respectively, within 2 days prior to sampling and/or vaccination of dams against gram-negative bacteria. Diarrhea was observed in 28.8% (95% CI, 24.7-32.8%) of the younger calves and 11.7% (95% CI, 9.1-15.5%) of the older calves. Importantly, Cryptosporidium spp. (odds ratio (OR) = 5.7; 95% CI, 3.3-9.9; p < 0.0001) and RVA (OR = 2.5; 95% CI, 1.2-5.1; p < 0.05) were both found to be risk factors for diarrhea in calves younger than 20 days old. Based on its high prevalence and OR, our results strongly suggest that Cryptosporidium spp. is the principal causative factor for diarrhea in the group of neonatal calves, whereas RVA seems to play a secondary role in the etiology of diarrhea in the studied farms, with about three-times lower prevalence and a half as high OR. Furthermore, a coinfection rate of Cryptosporidium spp. and RVA of 3.7% was observed in the group of younger calves, which strengthens the assumption that these events are independent. In contrast, due to a low infection rate of enteropathogens in older calves, mixed infection (<< 1%) was virtually absent in this group.

Molecular engineering improves antigen quality and enables integrated manufacturing of a trivalent subunit vaccine candidate for rotavirus.

BACKGROUND: Vaccines comprising recombinant subunit proteins are well-suited to low-cost and high-volume production for global use. The design of manufacturing processes to produce subunit vaccines depends, however, on the inherent biophysical traits presented by an individual antigen of interest. New candidate antigens typically require developing custom processes for each one and may require unique steps to ensure sufficient yields without product-related variants. RESULTS: We describe a holistic approach for the molecular design of recombinant protein antigens-considering both their manufacturability and antigenicity-informed by bioinformatic analyses such as RNA-seq, ribosome profiling, and sequence-based prediction tools. We demonstrate this approach by engineering the product sequences of a trivalent non-replicating rotavirus vaccine (NRRV) candidate to improve titers and mitigate product variants caused by N-terminal truncation, hypermannosylation, and aggregation. The three engineered NRRV antigens retained their original antigenicity and immunogenicity, while their improved manufacturability enabled concomitant production and purification of all three serotypes in a single, end-to-end perfusion-based process using the biotechnical yeast Komagataella phaffii. CONCLUSIONS: This study demonstrates that molecular engineering of subunit antigens using advanced genomic methods can facilitate their manufacturing in continuous production. Such capabilities have potential to lower the cost and volumetric requirements in manufacturing vaccines based on recombinant protein subunits.

Molecular characterization of group A rotavirus strains detected in alpacas (Vicugna pacos) from Peru.

The alpaca is a very important social and economic resource for the production of fibre and meat for Andean communities. Peru is the main producer of alpacas. Group A rotavirus (RVA) has been sporadically detected in alpacas. In this study, a total of 1423 faecal samples from alpacas from different locations of the Puno department in Peru were collected and analysed by an antigen-capture ELISA in order to detect RVA. Four per cent of the samples were RVA-positive (57/1423). The genotype constellation of three selected alpaca RVA strains were G3/8 P[1/14]-I2-R2/5-C2/3-M2/3-A17-N2/3-T6-E3-H3. Two of the analysed strains presented a bovine-like genotype constellation, whereas the third strain presented six segments belonging to the AU-1-like genogroup (G3, M3, C3, N3, T3 and E3), suggesting reassorting events. Monitoring of the sanitary health of juvenile alpacas is essential to reduce the rates of neonatal mortality and for the development of preventive health strategies.

A new passive immune strategy based on IgY antibodies as a key element to control neonatal calf diarrhea in dairy farms.

BACKGROUND: Neonatal diarrhea remains one of the main causes of morbi-mortality in dairy calves under artificial rearing. It is often caused by infectious agents of viral, bacterial, or parasitic origin. Cows vaccination and colostrum intake by calves during the first 6 h of life are critical strategies to prevent severe diarrhea but these are still insufficient. Here we report the field evaluation of a product based on IgY antibodies against group A rotavirus (RVA), coronavirus (CoV), enterotoxigenic Escherichia coli, and Salmonella sp. This product, named IgY DNT, has been designed as a complementary passive immunization strategy to prevent neonatal calf diarrhea. The quality of the product depends on the titers of specific IgY antibodies to each antigen evaluated by ELISA. In the case of the viral antigens, ELISA antibody (Ab) titers are correlated with protection against infection in calves experimentally challenged with RVA and CoV (Bok M, et al., Passive immunity to control bovine coronavirus diarrhea in a dairy herd in Argentina, 2017), (Vega C, et al., Vet Immunol Immunopathol, 142:156-69, 2011), (Vega C, et al., Res Vet Sci, 103:1-10, 2015). To evaluate the efficiency in dairy farms, thirty newborn Holstein calves were randomly assigned to IgY DNT or control groups and treatment initiated after colostrum intake and gut closure. Calves in the IgY DNT group received 20 g of the oral passive treatment in 2 L of milk twice a day during the first 2 weeks of life. Animals were followed until 3 weeks of age and diarrhea due to natural exposure to infectious agents was recorded during all the experimental time. RESULTS: Results demonstrate that the oral administration of IgY DNT during the first 2 weeks of life to newborn calves caused a delay in diarrhea onset and significantly reduced its severity and duration compared with untreated calves. Animals treated with IgY DNT showed a trend towards a delay in RVA infection with significantly shorter duration and virus shedding compared to control calves. CONCLUSIONS: This indicates that IgY DNT is an effective product to complement current preventive strategies against neonatal calf diarrhea in dairy farms. Furthermore, to our knowledge, this is the only biological product available for the prevention of virus-associated neonatal calf diarrhea.

Nanoanticuerpos derivados de llamas: una alternativa innovadora para controlar las diarreas por rotavirus y norovirus humano / Llama-derived nanobodies: an innovative approach to control human rotavirus and norovirus diarrhea

Las diarreas agudas son una de las afecciones más comunes en personas de todas las edades, especialmente en niños menores de 5 años. Rotavirus (RV) es la principal causa de diarrea en niños a nivel mundial seguido por norovirus (NoV). Sin embargo, a partir de la implementación de la vacunación con RV, NoV pasó a tener un rol preponderante en varios países como EEUU y Nicaragua. En Argentina, luego del inicio de la vacunación obligatoria de RV se logró un alto porcentaje de cobertura vacunal y se evidenció un descenso del 10% en la ocurrencia de diarreas agudas en general y un descenso del 50% de las diarreas causadas específicamente por RV, demostrando el éxito rotundo de la intervención. No existen al momento vacunas contra NoV, lo que lleva al desarrollo de otras estrategias de contención. A través del laboratorio INCUINTA del Instituto Nacional de Tecnología Agropecuaria (INTA), se han desarrollado y patentado nanoanticuerpos VHH contra RV y NoV con propiedades que los transforman en excelentes herramientas para el desarrollo de métodos de diagnóstico y para su utilización en estrategias de inmunidad pasiva oral para el tratamiento y prevención de la infección causada por estos agentes virales. Los nanoanticuerpos anti RV fueron utilizados con éxito para el desarrollo de un kit de ELISA nacional validado junto con el Instituto Malbrán que está siendo registrado para su comercialización. Además, demostraron poseer capacidad neutralizante de la infección contra RV in vitro y protección frente a la diarrea y excreción viralen los modelos de ratones lactantes y cerdos gnotobióticos. Los nano anticueroscontra NoV poseen alta afinidad para la detección del virus y fueron capaces de bloquear la unión del virus a los receptores celulares in vitro, demostrando su potencial como moléculas terapéuticas y de diagnóstico. El objetivo final de este desarrollo es completar la triada diagnostico-prevencióntratamiento para el control de las gastroenteritis virales con una tecnología de vanguardia de industria nacional.

Acute diarrhea is a common affection in people ofall ages, especially in children under five years old. Rotavirus (RV) is the major cause of diarrhea in children worldwide followed by norovirus (NoV). However, afterthe introduction of RV vaccine in the vaccination calendar in several countries like the USA and Nicaragua, NoV is having an important role in gastroenteritis incidence. In Argentina, after the introduction of the RV vaccine, high vaccine coverage was reached and the overall acute diarrhea incidence was reduced in a 10% and RV-associated diarrhea was reduced 50%, leading to complete success of the vaccination strategy. At the moment, there are no vaccines against NoV, thus, it is a need to develop therapies to control this disease. INCUINTA has developed and patented two sets of VHH nanobodies against RV and NoV with unique properties that make them suitable for the development of diagnostic tools, as well as to design oral passive immune strategies to preventand treat these two viral infections associated with diarrhea. VHH against rotavirus were able to neutralize RV infection in a suckling mouse model and in a gnotobiotic pig model of human RV infection and disease. Also, they were used to develop an ELISA kit for RV detection in human and animal samples. The ELISA kit was statistically validated in collaboration with the Malbrán Institute and is being registered for commercialization. VHH to NoVs showed high affinity to the virus and blocked the interaction between the virus and histo-blood antigens in target cells in vitro, showing to be a potential tool to detect and treat NoV infection. The final goal of this project is to complete the diagnostic-prevention-treatment triad to control viral gastroenteritis using a vanguard technology made in Argentina.

Passive immunity to control Bovine coronavirus diarrhea in a dairy herd in Argentina.

Bovine coronavirus (BCoV) is a viral enteric pathogen associated with calf diarrhea worldwide being, in Argentina, mostly detected in dairy husbandry systems. The aim of the present work was to study if maternal IgG1 antibodies (Abs) to BCoV acquired by colostrum intake modulate the development of BCoV infection in calves reared in a dairy farm in Argentina. Thirty Holstein calves were monitored during their first 60 days of age. Animals were classified into two groups depending on their initial BCoV IgG1 Ab titers. The "failure of passive transfer" (FPT) group had significantly lower IgG1 Abs to BCoV than the "acceptable passive transfer" (APT) group of calves (log10 1.98 vs. 3.38 respectively) (p<0.0001). These differences were also observed when the total protein levels in both groups were compared (p=0.0081). Moreover, 71% (5/7) of calves from the FPT group showed IgG1 seroconversion to BCoV compared to 29.4% (5/17) of animals from the APT group. Regarding viral circulation, BCoV was detected in 10% (3/30) of all calves and BCoV IgG1 Ab seroconversion was detected in 42% of the total animals showing that almost half of the calves were infected with BCoV. In conclusion, calves with high titers of specific BCoV IgG1 (≥1024) were mostly protected against viral infection, while animals with low titers of IgG1 (<1024) were mostly infected with BCoV. IgG1 Abs from colostrum origin are critical for prevention of BCoV infection.

Molecular detection of bovine Noroviruses in Argentinean dairy calves: Circulation of a tentative new genotype.

Bovine noroviruses are enteric pathogens detected in fecal samples of both diarrheic and non-diarrheic calves from several countries worldwide. However, epidemiological information regarding bovine noroviruses is still lacking for many important cattle producing countries from South America. In this study, three bovine norovirus genogroup III sequences were determined by conventional RT-PCR and Sanger sequencing in feces from diarrheic dairy calves from Argentina (B4836, B4848, and B4881, all collected in 2012). Phylogenetic studies based on a partial coding region for the RNA-dependent RNA polymerase (RdRp, 503 nucleotides) of these three samples suggested that two of them (B4836 and B4881) belong to genotype 2 (GIII.2) while the third one (B4848) was more closely related to genotype 1 (GIII.1) strains. By deep sequencing, the capsid region from two of these strains could be determined. This confirmed the circulation of genotype 1 (B4848) together with the presence of another sequence (B4881) sharing its highest genetic relatedness with genotype 1, but sufficiently distant to constitute a new genotype. This latter strain was shown in silico to be a recombinant: phylogenetic divergence was detected between its RNA-dependent RNA polymerase coding sequence (genotype GIII.2) and its capsid protein coding sequence (genotype GIII.1 or a potential norovirus genotype). According to this data, this strain could be the second genotype GIII.2_GIII.1 bovine norovirus recombinant described in literature worldwide. Further analysis suggested that this strain could even be a potential norovirus GIII genotype, tentatively named GIII.4. The data provides important epidemiological and evolutionary information on bovine noroviruses circulating in South America.

Llama nanoantibodies with therapeutic potential against human norovirus diarrhea.

Noroviruses are a major cause of acute gastroenteritis, but no vaccines or therapeutic drugs are available. Llama-derived single chain antibody fragments (also called VHH) are small, recombinant monoclonal antibodies of 15 kDa with several advantages over conventional antibodies. The aim of this study was to generate recombinant monoclonal VHH specific for the two major norovirus (NoV) genogroups (GI and GII) in order to investigate their potential as immunotherapy for the treatment of NoV diarrhea. To accomplish this objective, two llamas were immunized with either GI.1 (Norwalk-1968) or GII.4 (MD2004) VLPs. After immunization, peripheral blood lymphocytes were collected and used to generate two VHH libraries. Using phage display technology, 10 VHH clones specific for GI.1, and 8 specific for GII.4 were selected for further characterization. All VHH recognized conformational epitopes in the P domain of the immunizing VP1 capsid protein, with the exception of one GII.4 VHH that recognized a linear P domain epitope. The GI.1 VHHs were highly specific for the immunizing GI.1 genotype, with only one VHH cross-reacting with GI.3 genotype. The GII.4 VHHs reacted with the immunizing GII.4 strain and showed a varying reactivity profile among different GII genotypes. One VHH specific for GI.1 and three specific for GII.4 could block the binding of homologous VLPs to synthetic HBGA carbohydrates, saliva, and pig gastric mucin, and in addition, could inhibit the hemagglutination of red blood cells by homologous VLPs. The ability of Nov-specific VHHs to perform well in these surrogate neutralization assays supports their further development as immunotherapy for NoV treatment and immunoprophylaxis.

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.

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 pigs 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.