Caveats of chimpanzee ChAdOx1 adenovirus-vectored vaccines to boost anti-SARS-CoV-2 protective immunity in mice.

Several COVID-19 vaccines use adenovirus vectors to deliver the SARS-CoV-2 spike (S) protein. Immunization with these vaccines promotes immunity against the S protein, but against also the adenovirus itself. This could interfere with the entry of the vaccine into the cell, reducing its efficacy. Herein, we evaluate the efficiency of an adenovirus-vectored vaccine (chimpanzee ChAdOx1 adenovirus, AZD1222) in boosting the specific immunity compared to that induced by a recombinant receptor-binding domain (RBD)-based vaccine without viral vector. Mice immunized with the AZD1222 human vaccine were given a booster 6 months later, with either the homologous vaccine or a recombinant vaccine based on RBD of the delta variant, which was prevalent at the start of this study. A significant increase in anti-RBD antibody levels was observed in rRBD-boosted mice (31-61%) compared to those receiving two doses of AZD1222 (0%). Significantly higher rates of PepMix™- or RBD-elicited proliferation were also observed in IFNγ-producing CD4 and CD8 cells from mice boosted with one or two doses of RBD, respectively. The lower efficiency of the ChAdOx1-S vaccine in boosting specific immunity could be the result of a pre-existing anti-vector immunity, induced by increased levels of anti-adenovirus antibodies found both in mice and humans. Taken together, these results point to the importance of avoiding the recurrent use of the same adenovirus vector in individuals with immunity and memory against them. It also illustrates the disadvantages of ChAdOx1 adenovirus-vectored vaccine with respect to recombinant protein vaccines, which can be used without restriction in vaccine-booster programs. KEY POINTS: • ChAdOx1 adenovirus vaccine (AZD1222) may not be effective in boosting anti-SARS-CoV-2 immunity • A recombinant RBD protein vaccine is effective in boosting anti-SARS-CoV-2 immunity in mice • Antibodies elicited by the rRBD-delta vaccine persisted for up to 3 months in mice.

Towards the development of an epitope-focused vaccine for SARS-CoV-2.

The rapid spread of COVID-19 on all continents and the mortality induced by SARS-CoV-2 virus, the cause of the pandemic coronavirus disease 2019 (COVID-19) has motivated an unprecedented effort for vaccine development. Inactivated viruses as well as vaccines focused on the partial or total sequence of the Spike protein using different novel platforms such us RNA, DNA, proteins, and non-replicating viral vectors have been developed. The high global need for vaccines, now and in the future, and the emergence of new variants of concern still requires development of accessible vaccines that can be adapted according to the most prevalent variants in the respective regions. Here, we describe the immunogenic properties of a group of theoretically predicted RBD peptides to be used as the first step towards the development of an effective, safe and low-cost epitope-focused vaccine. One of the tested peptides named P5, proved to be safe and immunogenic. Subcutaneous administration of the peptide, formulated with alumina, induced high levels of specific IgG antibodies in mice and hamsters, as well as an increase of IFN-γ expression by CD8+ T cells in C57 and BALB/c mice upon in vitro stimulation with P5. Neutralizing titers of anti-P5 antibodies, however, were disappointingly low, a deficiency that we will attempt to resolve by the inclusion of additional immunogenic epitopes to P5. The safety and immunogenicity data reported in this study support the use of this peptide as a starting point for the design of an epitope restricted vaccine.

Heparan Sulfate and Sialic Acid in Viral Attachment: Two Sides of the Same Coin?

Sialic acids and heparan sulfates make up the outermost part of the cell membrane and the extracellular matrix. Both structures are characterized by being negatively charged, serving as receptors for various pathogens, and are highly expressed in the respiratory and digestive tracts. Numerous viruses use heparan sulfates as receptors to infect cells; in this group are HSV, HPV, and SARS-CoV-2. Other viruses require the cell to express sialic acids, as is the case in influenza A viruses and adenoviruses. This review aims to present, in a general way, the participation of glycoconjugates in viral entry, and therapeutic strategies focused on inhibiting the interaction between the virus and the glycoconjugates. Interestingly, there are few studies that suggest the participation of both glycoconjugates in the viruses addressed here. Considering the biological redundancy that exists between heparan sulfates and sialic acids, we propose that it is important to jointly evaluate and design strategies that contemplate inhibiting the interactions of both glycoconjugates. This approach will allow identifying new receptors and lead to a deeper understanding of interspecies transmission.

An RBD-Based Diagnostic Method Useful for the Surveillance of Protective Immunity against SARS-CoV-2 in the Population.

After more than two years, the COVID-19 pandemic is still ongoing and evolving all over the world; human herd immunity against SARS-CoV-2 increases either by infection or by unprecedented mass vaccination. A substantial change in population immunity is expected to contribute to the control of transmission. It is essential to monitor the extension and duration of the population's immunity to support the decisions of health authorities in each region and country, directed to chart the progressive return to normality. For this purpose, the availability of simple and cheap methods to monitor the levels of relevant antibodies in the population is a widespread necessity. Here, we describe the development of an RBD-based ELISA for the detection of specific antibodies in large numbers of samples. The recombinant expression of an RBD-poly-His fragment was carried out using either bacterial or eukaryotic cells in in vitro culture. After affinity chromatography purification, the performance of both recombinant products was compared by ELISA in similar trials. Our results showed that eukaryotic RBD increased the sensitivity of the assay. Interestingly, our results also support a correlation of the eukaryotic RBD-based ELISA with other assays aimed to test for neutralizing antibodies, which suggests that it provides an indication of protective immunity against SARS-CoV-2.

Immunogenic and antigenic analysis of recombinant NSP1 and NSP11 of PRRS virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus in the order Nidovirales, family Arteriviridae, genus Betaarterivirus. Antibodies against nonstructural proteins (NSPs) from this virus can be found in pigs starting 4 days postinfection and they remain detectable for several months. OBJECTIVE: The goal of this study was to evaluate the immunogenicity and antigenic properties of recombinant proteins NSP1 and NSP11 expressed in Escherichia coli cells, as well as to assess the neutralization activity that they elicit. METHODS: We obtained the complete ORF-1 genes coding for NSP1 and NSP11 from PRRSV using the VR-2332 strain. Cloning was performed with the pET23a(+) vector with a histidine tag (His6), linearized by restriction enzyme digestion; the expression of the NSP1 and NSP11 clones was induced in OverExpress C41(DE3) chemically competent cells. Recombinant proteins were used to generate hyperimmune sera and we perform serological assays to confirm neutralizing antibodies. RESULTS: The expressed recombinant NSP1 and NSP11 were found to be immunogenic when injected in pigs, as well as demonstrated higher specificity in recognition of antigen in field sera from pigs positive infected with PRRSV. Furthermore, both NSP1 and NSP11 recombinant proteins elicited PRRSV neutralizing antibodies. CONCLUSIONS: In this study, we demonstrated the immune humoral response to NSP 1 and NSP11, and neutralizing-antibody response to PRRSV VR2332 strain in sera from hyperimmunized pigs.

Molecular identification and phylogenetic characterization of influenza A virus at a wildlife-livestock interface in Mexico.

Influenza A virus (IAV) outbreaks constitute a constant threat to public health and pose a remarkable impact on socio-economic systems worldwide. Interactions between wild and domestic birds, humans and swine can lead to spillover events. Backyard livestock systems in proximity to wetlands represent high-risk areas for viral spread. However, some gaps remain in our knowledge of IAV transmission at the wildlife-livestock interface in Mexico. Hence, the study aimed at molecular identification and phylogenetic characterization of IAV in the wild duck-backyard livestock interface at a wetland of Mexico. A total of 875 animals were tested by real-time RT-PCR (qRT-PCR). We detected IAV in 3.68% of the wild ducks sampled during the winter season 2016-2017. Nonetheless, the samples obtained from backyard poultry and swine tested negative. The highest IAV frequency (11.10%) was found in the Mexican duck (Anas diazi). Subtypes H1N1, H3N2 and H5N2 were detected. Phylogenetic analyses revealed that IAV detected in wild birds from the Lerma wetlands was mostly related to swine and poultry IAV strains previously isolated in the United States and Mexico. Except, the UIFMVZ377/H5N2 related to North American waterbirds. In conclusion, the co-circulation of three IAV subtypes in wild ducks close to backyard farms in Mexico, as well as the local identification of influenza viruses genetically related to Mexican and North American IAV strains, highlights the importance of the Lerma marshes for influenza surveillance given the close interaction among wild birds, poultry, pigs and humans.

Eco-Epidemiological Evidence of the Transmission of Avian and Human Influenza A Viruses in Wild Pigs in Campeche, Mexico.

Influenza, a zoonosis caused by various influenza A virus subtypes, affects a wide range of species, including humans. Pig cells express both sialyl-α-2,3-Gal and sialyl-α-2,6-Gal receptors, which make them susceptible to infection by avian and human viruses, respectively. To date, it is not known whether wild pigs in Mexico are affected by influenza virus subtypes, nor whether this would make them a potential risk of influenza transmission to humans. In this work, 61 hogs from two municipalities in Campeche, Mexico, were sampled. Hemagglutination inhibition assays were performed in 61 serum samples, and positive results were found for human H1N1 (11.47%), swine H1N1 (8.19%), and avian H5N2 (1.63%) virus variants. qRT-PCR assays were performed on the nasal swab, tracheal, and lung samples, and 19.67% of all hogs were positive to these assays. An avian H5N2 virus, first reported in 1994, was identified by sequencing. Our results demonstrate that wild pigs are participating in the exposure, transmission, maintenance, and possible diversification of influenza viruses in fragmented habitats, highlighting the synanthropic behavior of this species, which has been poorly studied in Mexico.

First report and phylogenetic analysis of porcine deltacoronavirus in Mexico.

Porcine deltacoronavirus has caused great economic losses in the swine industry worldwide. In this study, we carried out the first detection, sequencing and characterization of this virus in Mexico. We analysed 885 rectal samples by multiplex RT-PCR to determine coinfections. In addition, the Spike gene was amplified, sequenced and analysed phylogenetically. We found 85 positive samples for porcine deltacoronavirus, representing 9.6% of the total samples, and we determined that the most frequent coinfection was with porcine epidemic diarrhoea virus (54.1%). Four sequences of Mexican isolates were most closely related to those of the United States. The antigenic regions and the glycosylation site of the strains obtained coincide with those previously reported. This relationship is probably related to the commercial exchange of pigs between the US and Mexico and the geographical proximity of these two countries.

Preclinical evidences of safety of a new synthetic adjuvant to formulate with the influenza human vaccine: absence of subchronic toxicity and mutagenicity.

Context: Influenza is a severe, life-threatening viral disease that can be prevented by vaccination. However, the anti-influenza human vaccine failed to show the required efficacy both in infants under 5 years old and in the elder population, who are among those with the highest risk of developing severe complications after influenza infection. Therefore, it is of high importance to improve the vaccine efficacy and ensure its safety in these susceptible populations. GK-1, a novel 18-aa peptide adjuvant, has been proved to increase the immunogenicity of the human influenza vaccine in both young and aged mice. Objective: A preclinical study of the toxicity profile of GK-1 following the World Health Organization guidelines to support its use was herein conducted. Material and methods: GK-1 was synthetically produced following Good Manufacturing Practices. The toxicological evaluation of GK-1 peptide was performed in rats after repeated dose-ranging trials by the subcutaneous route. The mutagenic potential of GK-1 was assessed by the micronucleus, chromosomal aberration, and Ames tests, in accordance with OECD Guidelines. Results: GK-1 did not show toxic effects at doses up to 12.5mg/kg, corresponding to 25 times the dose intended for human use. No indications of mutagenic potential were observed. GK-1 after dermal administration was well tolerated locally. Conclusion: The efficacy of GK-1 to improve influenza vaccine protection, along with the absence of toxicity and mutagenicity, as reported herein, support the evaluation of this peptide in a clinical trial as a novel adjuvant for human use.

Identification and genomic characterization of influenza viruses with different origin in Mexican pigs.

Swine influenza is a worldwide disease, which causes damage to the respiratory system of pigs. The H1N1 and H3N2 subtypes circulate mainly in the swine population of Mexico. There is evidence that new subtypes of influenza virus have evolved genetically and have been rearranged with human viruses and from other species; therefore, the aim of our study was to identify and characterize the genetic changes that have been generated in the different subtypes of the swine influenza virus in Mexican pigs. By sequencing and analyzing phylogenetically the eight segments that form the virus genome, the following subtypes were identified: H1N1, H3N2, H1N2 and H5N2; of which, a H1N1 subtype had a high genetic relationship with the human influenza virus. In addition, a H1N2 subtype related to the porcine H1N2 virus reported in the United States was identified, as well as, two other viruses of avian origin from the H5N2 subtype. Particularly for the H5N2 subtype, this is the first time that its presence has been reported in Mexican pigs. The analysis of these sequences demonstrates that in the swine population of Mexico, circulate viruses that have suffered punctual-specific mutations and rearrangements of their proteins with different subtypes, which have successfully adapted to the Mexican swine population.

High stability of the immunomodulatory GK-1 synthetic peptide measured by a reversed phase high-performance liquid chromatography method.

The 18-mer anionic peptide GK-1 has been successfully employed to improve the immunogenicity and protective response induced by the influenza vaccine and exhibited some degree of protection against experimental murine melanoma. In this study, a sensitive and quantitative reversed-phase HPLC method was developed to study GK-1 stability under different pH, temperature and storage time. The analysis was carried out on a Sunfire C18 column with mobile phase of acetonitrile-water containing 0.02% TFA. The detection was performed on an UV/Vis Detector at 220nm. The method was validated with respect to linearity, limits of detection and quantification, precision and selectivity. The linear calibration curves were obtained in the concentration range of 0.015-0.24mg/mL (r2=0.99) with lower limits of detection (LOD) and quantification (LOQ) of 0.001 and 0.018mg/mL, respectively. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 3% at all quality control levels. Forced degradation studies were conducted by introducing a sample of GK-1 peptide standard solution to different conditions of pH (from 2 to 8), temperature (4, 25 and 40°C) and storage time (10days to 6 months). The peptide GK-1 showed to be stable under different ranges of pH and temperature; however it was susceptible to prolonged storage at room temperature. Results shown in this study sustain the high stability of the GK-1 peptide using a reliable new selective and precise method suitable for its analysis.

Transgenic papaya: a useful platform for oral vaccines.

MAIN CONCLUSION: Transgenic papaya callus lines expressing the components of the S3Pvac vaccine constitute a stable platform to produce an oral vaccine against cysticercosis caused by Taenia solium or T. crassiceps. The development of effective delivery systems to cope with the reduced immunogenicity of new subunit vaccines is a priority in vaccinology. Herein, experimental evidence supporting a papaya-based platform to produce needle-free, recombinant, highly immunogenic vaccines is shown. Papaya (Carica papaya) callus lines were previously engineered by particle bombardment to express the three protective peptides of the S3Pvac anti-cysticercosis vaccine (KETc7, KETc12, KETc1). Calli were propagated in vitro, and a stable integration and expression of the target genes has been maintained, as confirmed by PCR, qRT-PCR, and HPLC. These results point papaya calli as a suitable platform for long-term transgenic expression of the vaccine peptides. The previously demonstrated protective immunogenic efficacy of S3Pvac-papaya orally administered to mice is herein confirmed in a wider dose-range and formulated with different delivery vehicles, adequate for oral vaccination. This protection is accompanied by an increase in anti-S3Pvac antibody titers and a delayed hypersensitivity response against the vaccine. A significant increase in CD4+ and CD8+ lymphocyte proliferation was induced in vitro by each vaccine peptide in mice immunized with the lowest dose of S3Pvac papaya (0.56 ng of the three peptides in 0.1 µg of papaya callus total protein per mouse). In pigs, the obliged intermediate host for Taenia solium, S3Pvac papaya was also immunogenic when orally administered in a two-log dose range. Vaccinated pigs significantly increased anti-vaccine antibodies and mononuclear cell proliferation. Overall, the oral immunogenicity of this stable S3Pvac-papaya vaccine in mice and pigs, not requiring additional adjuvants, supports the interest in papaya callus as a useful platform for plant-based vaccines.

Towards identification of the mechanisms of action of parasite-derived peptide GK1 on the immunogenicity of an influenza vaccine.

Previous studies have shown that the synthetic peptide GK1, derived from Taenia crassiceps cysticerci, enhances the immunogenicity of the commercial inactivated influenza vaccine Fluzone in both young and aged mice. In particular, antibody responses were much improved. Since GK1 is a peptide and is rapidly cleared from the body, it offers the possibility to improve vaccine performance without undesirable effects. This study was therefore designed to understand the mechanisms of action involved in the adjuvant properties of GK1. For this, transgenic mice expressing a T-cell receptor specific for an epitope from the influenza virus hemagglutinin (HA) protein were employed. The GK1 peptide significantly increased the in vivo proliferative response of HA-specific CD4+ T cells when it was coimmunized with the HA epitope. Dendritic cells treated in vitro with GK1 were capable of enhancing T-cell activation. Furthermore, in synergy with lipopolysaccharide, GK1 enhanced the expression of major histocompatibility complex class II and costimulatory molecules of dendritic cells and promoted the secretion of proinflammatory cytokines and chemokines upon antigen-driven T-cell interaction. These data provide important insights into the mechanism that underlies the GK1 adjuvant capacity observed previously and underline the feasibility of using the transgenic mouse model described herein as a tool for investigation of the modes of action of different influenza vaccine adjuvants.