Oral administration of a recombinant modified RBD antigen of SARS-CoV-2 as a possible immunostimulant for the care of COVID-19.

BACKGROUND: Developing effective vaccines against SARS-CoV-2 that consider manufacturing limitations, equitable access, and acceptance is necessary for developing platforms to produce antigens that can be efficiently presented for generating neutralizing antibodies and as a model for new vaccines. RESULTS: This work presents the development of an applicable technology through the oral administration of the SARS-CoV-2 RBD antigen fused with a peptide to improve its antigenic presentation. We focused on the development and production of the recombinant receptor binding domain (RBD) produced in E. coli modified with the addition of amino acids extension designed to improve antigen presentation. The production was carried out in shake flask and bioreactor cultures, obtaining around 200 mg/L of the antigen. The peptide-fused RBD and peptide-free RBD proteins were characterized and compared using SDS-PAGE gel, high-performance chromatography, and circular dichroism. The peptide-fused RBD was formulated in an oil-in-water emulsion for oral mice immunization. The peptide-fused RBD, compared to RBD, induced robust IgG production in mice, capable of recognizing the recombinant RBD in Enzyme-linked immunosorbent assays. In addition, the peptide-fused RBD generated neutralizing antibodies in the sera of the dosed mice. The formulation showed no reactive episodes and no changes in temperature or vomiting. CONCLUSIONS: Our study demonstrated the effectiveness of the designed peptide added to the RBD to improve antigen immunostimulation by oral administration.

Multiplex assay for the simultaneous detection of antibodies against small ruminant lentivirus, Mycobacterium avium subsp. paratuberculosis, and Brucella melitensis in goats.

Background and Aim: Brucellosis, paratuberculosis (PTb), and infections caused by small ruminant lentivirus (SRLV), formerly known as caprine arthritis encephalitis virus (CAEV), adversely affect goat production systems. Nonetheless, commonly used diagnostic tests can only determine one analyte at a time, increasing disease surveillance costs, and limiting their routine use. This study aimed to design and validate a multiplex assay for antibody detection against these three diseases simultaneously. Materials and Methods: Two recombinant proteins from the SRLV (p16 and gp38), the native hapten of Brucella melitensis, and the paratuberculosis-protoplasmic antigen 3 from Mycobacterium avium subsp. paratuberculosis (MAP) were used to devise and assess a multiplex assay. Conditions for the Luminex® multiplex test were established and validated by sensitivity, specificity, repeatability, and reproducibility parameters. Cut-off points for each antigen were also established. Results: The 3-plex assay had high sensitivity (84%) and specificity (95%). The maximum coefficients of variation were 23.8% and 20.5% for negative and positive control samples, respectively. The p16 and gp38 SRLV antigens are 97% and 95%, similar to the CAEV sequence found in GenBank, respectively. Conclusion: The multiplex test can be effectively used for the simultaneous detection of antibodies against SRLV, MAP and B. melitensis in goats.

Altered neutrophil-to-lymphocyte ratio in sepsis secondary to canine parvoviral enteritis treated with and without an immunomodulator in puppies.

Neutrophil-to-lymphocyte ratio (NLR) is a cheap and easy-to-obtain biomarker that mirrors the balance between innate and adaptive immunity. Cortisol and catecholamines have been identified as major drivers of NLR. High cortisol levels increase neutrophils while simultaneously decreasing lymphocyte counts. Likewise, endogenous catecholamines may cause leukocytosis and lymphopenia. Thus, NLR allows us to monitor patient severity in conditions such as sepsis. Twenty-six puppies with sepsis secondary to canine parvoviral enteritis were treated with and without an immunomodulator. Our group determined the NLR and the plasmatic cortisol levels by chemiluminescence, and norepinephrine (NE) and epinephrine (E) by HPLC during the first 72 h of clinical follow-up. Our results showed that at admission puppies presented an NLR value of 1.8, cortisol of 314.9 nmol/L, NE 3.7, and E 3.3 pmol/mL. Both treatments decreased admission NLR values after 24 h of treatment. However, only the puppies treated with the immunomodulator (I) remained without significant changes in NLR (0.7-1.4) compared to the CT group, and that showed a significant difference (P < 0.01) in their NLR value (0.4-4.6). In addition, we found significant differences in the slope values between the admission and final values of NLR (P < 0.005), cortisol (P < 0.02), and E (P < 0.05) between treatments. Then, our data suggest that the immunomodulator positively affects the number of lymphocytes and neutrophils involved in NLR as well as major drivers like cortisol and epinephrine, which is reflected in clinical parameters and survival.

Increased survival in puppies affected by Canine Parvovirus type II using an immunomodulator as a therapeutic aid.

Canine parvovirus type II (CPV-2) infection induces canine parvoviral enteritis (CPE), which in turn promotes sepsis and systemic inflammatory response syndrome (SIRS). Mortality in this disease is usually registered within 48-72 h post-hospitalization, the critical period of the illness. It has been recently described that the use of an immunomodulator, whose major component is monomeric ubiquitin (mUb) without the last two glycine residues (Ub∆GG), in pediatric human patients with sepsis augments survival. It is known that CXCR4 is the cell receptor of extracellular ubiquitin in humans. This work aimed to explore the effect of one immunomodulator (human Dialyzable Leukocyte Extract-hDLE) as a therapeutic auxiliary in puppies with sepsis and SIRS induced by CPE. We studied two groups of puppies with CPV-2 infection confirmed by polymerase chain reaction. The first group received conventional treatment (CT) and vehicle (V), while the second group received CT plus the immunomodulator (I). We assessed both groups' survival, clinical condition, number of erythrocytes, neutrophils, and lymphocytes during the hospitalization period. In addition, hematocrit, hemoglobin, plasma proteins and cortisol values, as well as norepinephrine/epinephrine and serotonin concentration were determined. Puppies treated with CT + I showed 81% survival, mild clinical signs, and a significant decrease in circulating neutrophils and lymphocytes in the critical period of the treatment. In contrast, the CT + V group presented a survival of 42%, severe clinical status, and no improvement of the parameters evaluated in the critical period of the disease. We determined in silico that human Ub∆GG can bind to dog CXCR4. In conclusion, the administration of a human immunomodulator (0.5 mg/day × 5 days) to puppies with CPE under six months of age reduces the severity of clinical signs, increases survival, and modulates inflammatory cell parameters. Further studies are necessary to take full advantage of these clinical findings, which might be mediated by the human Ub∆GG to canine CXCR4 interaction.

Integrative overview of antibodies against SARS-CoV-2 and their possible applications in COVID-19 prophylaxis and treatment.

SARS-CoV-2 is a novel ß-coronavirus that caused the COVID-19 pandemic disease, which spread rapidly, infecting more than 134 million people, and killing almost 2.9 million thus far. Based on the urgent need for therapeutic and prophylactic strategies, the identification and characterization of antibodies has been accelerated, since they have been fundamental in treating other viral diseases. Here, we summarized in an integrative manner the present understanding of the immune response and physiopathology caused by SARS-CoV-2, including the activation of the humoral immune response in SARS-CoV-2 infection and therefore, the synthesis of antibodies. Furthermore, we also discussed about the antibodies that can be generated in COVID-19 convalescent sera and their associated clinical studies, including a detailed characterization of a variety of human antibodies and identification of antibodies from other sources, which have powerful neutralizing capacities. Accordingly, the development of effective treatments to mitigate COVID-19 is expected. Finally, we reviewed the challenges faced in producing potential therapeutic antibodies and nanobodies by cell factories at an industrial level while ensuring their quality, efficacy, and safety.

Valproic acid promotes a decrease in mycobacterial survival by enhancing nitric oxide production in macrophages stimulated with IFN-γ.

Tuberculosis is one of the leading causes of mortality worldwide, it is caused by Mycobacterium tuberculosis (Mtb), a bacteria that employs several strategies to evade the host immune response. For instance, Mtb interferes with the overexpression of class II transactivator (CIITA) in macrophages exposed to IFN-γ by inhibiting histone acetylation at its promoter, which can be reverted by the histone deacetylase inhibitor (HDACi) sodium butyrate. In this work, we evaluated whether a different HDACi, valproic acid (VPA), could revert the inhibition of gene expression induced by Mtb. J774 macrophages treated with VPA and IFN-γ unexpectedly induced a higher expression of the inducible nitric oxide synthase and a higher production of nitric oxide when exposed to the 19 kDa lipoprotein of Mtb or the whole bacteria. However, VPA was unable to revert the inhibition of CIITA expression induced by the 19 kDa lipoprotein of Mtb. Finally, macrophages infected with Mtb and treated with VPA and IFN-γ showed a significant reduction in intracellular bacteria. Our findings suggest a new therapeutic potential of VPA for the treatment of tuberculosis.

Vulvovaginitis y balanopostitis pustular sugerente a herpesvirus caprino-1 en cabras (Querétaro, México) / Pustular vulvovaginitis and balanoposthitis suggestive of caprine herpesvirus-1 infection in goats (Queretaro, Mexico)

Infectious pustular vulvovaginitis and balanoposthitis in goats is caused by caprine herpesvirus 1 (CpHV-1), from the Herpesviridae family Alphaherpesvirinae subfamily, sometimes produces abortion, neonatal enteritis and respiratory disease in adult goats. In a herd of 244 dairy and meat goats, physical examinations were performed in does and bucks, presenting vulvar and preputial lesions. Necropsies were done in four discarded animals (one male and two females) and one stillborn; immunohistochemistry (IHC) with bovine herpesvirus 1 (BHV-1) antibodies in prepuce and vulva samples was carried out. Penis and prepuce exfoliative cytology was carried out in six bucks. Prepuce biopsies were collected for electron microscopy and viral isolation. Macroscopically, moderate to severe coalescent multifocal pustular and ulcerative vulvovaginitis was observed; and severe coalescent multifocal pustular and ulcerative balanoposthitis. Microscopically, different degrees of inflammatory lesions with presence of eosinophilic and amphophilic intranuclear inclusion bodies were present. In electron microscopy, the prepuce samples and MBDK cell culture showed several intranuclear viral particles of 150 to 230 nm size. There was slight epithelial cell positivity in IHC with BHV-1 antibodies in vulva and prepuce histological cuts made at necropsy. In the viral isolate was observed a cytopathic effect and the presence of viral particles by microscopy electronic suggesting herpes virus. This is the first documented report suggesting the presence of herpes virus outbreak in caprine herds in Mexico.

La vulvovaginitis y balanopostitis pustular infecciosa en cabras es producida por herpesvirus caprino 1 (CpHV-1), de la familia Herpesviridae subfamilia Alphaherpesvirinae, que ocasionalmente produce aborto, enteritis neonatal y enfermedad respiratoria en las cabras adultas. En un rebaño de 244 cabras de genotipo lechero y cárnico; se realizaron exámenes físicos de hembras y sementales en los que se observaron lesiones vulvares y prepuciales. Se realizó la necropsia de cuatro animales adultos de desecho (dos hembras y un macho) y un neonato muerto; se realizó inmunohistoquímica (IHQ) con anticuerpos de herpesvirus bovino 1 (BHV1) en muestras de prepucio y vulva. Se analizaron las citologías exfoliativas de pene y prepucio de 6 sementales. Se recolectaron y analizaron biopsias de prepucio para microscopía electrónica y aislamiento viral. Macroscópicamente, en las hembras se observó vulvovaginitis pustular y ulcerativa de moderada a grave multifocal coalescente, además de balanopostitis pustular y ulcerativa grave multifocal coalescente en los machos. Microscópicamente, se presentaron lesiones inflamatorias en diferentes grados, con presencia de cuerpos de inclusión intranucleares eosinofílicos y amfofílicos. En microscopía electrónica, las muestras de prepucio y cultivo celular de MBDK presentaron numerosas partículas virales intranucleares de un tamaño de 150 a 230 nm. En los cortes histológicos de prepucio y vulva de las necropsias se detectó una ligera positividad en las células epiteliales con anticuerpos BHV-1. En el aislamiento viral se observó efecto citopático, y la presencia de partículas virales mediante microscopía electrónica sugerentes a herpes virus. Este es el primer informe documentado que sugiere la presencia de herpes virus en rebaños caprinos de México.

Spoligotype analysis of Mycobacterium bovis isolates from Northern México.

Bovine tuberculosis is still rife in Latin America, producing huge economic losses. There are very few studies of the way this disease is spread through this geographical region, particularly in countries that border those that are almost free of Mycobacterium bovis. In this work, we have analyzed the spacer oligonucleotide typing (spoligotype) patterns of M. bovis isolates from cattle at Ciudad Juárez, a Mexican city close to El Paso, Texas. Fifty-eight M. bovis isolates collected from a herd in Northern Mexico were studied by spoligotyping. Nine spoligotype patterns were observed in total. Two were predominant (SB0121 and SB0140) and accounted for 50% and 14% of the isolates, respectively. Six patterns were found to be already described in an international M. bovis spoligotype database, while the other three (SB0985, SB0986, and SB0987) were novel. Interestingly, none of the isolates corresponded to any other Mexican pattern previously reported. This is the first spoligotype analysis of M. bovis strains from a border city between Mexico and the United States. The necessity for further studies to formulate a better identification of M. bovis strains within, and its dissemination between, the two countries is discussed.