Characterization of Canine Peyer's Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing.

The oral route is effective and convenient for vaccine administration to stimulate a protective immune response. GALT plays a crucial role in mucosal immune responses, with Peyer's patches (PPs) serving as the primary site of induction. A comprehensive understanding of the structures and functions of these structures is crucial for enhancing vaccination strategies and comprehending disease mechanisms; nonetheless, our current knowledge of these structures in dogs remains incomplete. We performed immunofluorescence and flow cytometry studies on canine PPs to identify cell populations and structures. We also performed single-cell RNA sequencing (scRNA-seq) to investigate the immune cell subpopulations present in PPs at steady state in dogs. We generated and validated an Ab specifically targeting canine M cells, which will be a valuable tool for elucidating Ag trafficking into the GALT of dogs. Our findings will pave the way for future studies of canine mucosal immune responses to oral vaccination and enteropathies. Moreover, they add to the growing body of knowledge in canine immunology, further expanding our understanding of the complex immune system of dogs.

Mucosal Vaccination with Live Attenuated Bordetella bronchiseptica Protects against Challenge in Wistar Rats.

Bordetella bronchiseptica (Bb) is a Gram-negative bacterium responsible for canine infectious respiratory disease complex (CIRDC). Several vaccines targeting this pathogen are currently licensed for use in dogs, but their mechanism of action and the correlates of protection are not fully understood. To investigate this, we used a rat model to examine the immune responses induced and the protection conferred by a canine mucosal vaccine after challenge. Wistar rats were vaccinated orally or intranasally on D0 and D21 with a live attenuated Bb vaccine strain. At D35, the rats of all groups were inoculated with 103 CFU of a pathogenic strain of B. bronchiseptica. Animals vaccinated via either the intranasal or the oral route had Bb-specific IgG and IgM in their serum and Bb-specific IgA in nasal lavages. Bacterial load in the trachea, lung, and nasal lavages was lower in vaccinated animals than in non-vaccinated control animals. Interestingly, coughing improved in the group vaccinated intranasally, but not in the orally vaccinated or control group. These results suggest that mucosal vaccination can induce mucosal immune responses and provide protection against a Bb challenge. This study also highlights the advantages of a rat model as a tool for studying candidate vaccines and routes of administration for dogs.

In Vitro Growth, Receptor Usage and Pathogenesis of Feline Morbillivirus in the Natural Host.

Feline morbillivirus (FeMV) is a recently discovered virus belonging to the genus Morbillivirus of the virus family Paramyxoviridae. Often, the virus has been detected in urine of cats with a history of urinary disease and has a worldwide distribution. Currently, it is unclear which receptor the virus uses to enter the target cells. Furthermore, many aspects of FeMV biology in vivo, including tissue tropism, pathogenesis, and virus excretion in the natural host remain unclear. In this study we analyzed the replication of FeMV in various cell lines. Secondly, we tested if the presence of feline SLAMF1 (Signaling Lymphocytic Activation Molecule family 1/CD150, principal entry receptor for other members of the Morbillivirus genus) improved FeMV replication efficiency in vitro. Finally, to elucidate in vivo biology in cats, as a natural host for FeMV, we experimentally infected a group of cats and monitored clinical symptoms, viremia, and excretion of the virus during the course of 56 days. Our study showed that FeMV shares some features with other morbilliviruses like the use of the SLAMF1 receptor. For the first time, experimental infection of SPF cats showed that FeMV does not induce an acute clinical disease like other morbilliviruses but can induce lesions in the kidneys, including tubulointerstitial nephritis. Further investigations are needed to confirm the site and dynamics of replication of FeMV in the urinary tract and the longer-term impact of FeMV-induced lesions on the renal function. Whether FeMV infection can result in chronic kidney disease will require the monitoring of cats over a longer period.

Quantitative Real-Time PCR Assays for the Detection of Pathogenic Leptospira Species in Urine and Blood Samples in Canine Vaccine Clinical Studies: a Rapid Alternative to Classical Culture Methods.

Leptospirosis is a vaccine-preventable bacterial zoonotic disease caused by pathogenic Leptospira species. The efficacy of Leptospira canine vaccines is assessed by challenging vaccinated and control dogs with virulent serovars of Leptospira, followed by detection of Leptospira in blood and urine. We assessed the consistency between results obtained for urine and blood samples from clinical studies with species-specific real-time quantitative PCR (qPCR) targeting the lipL32 gene and those obtained with the reference culture method. The specificity of the qPCR assay was confirmed by negative results for nonpathogenic Leptospira and for several canine viruses, bacteria, and parasites. The results from the two methods were compared using McNemar's test, kappa coefficient (κ), and percentage of agreement analyses. The results for numbers of positive and negative dogs were similar, with no false-negative results with the qPCR assay. For both blood and urine, there was strong agreement between the culture method and qPCR results (κ = 0.68 [95% confidence interval (CI), 0.62 to 0.74] and κ = 0.65 [95% CI, 0.59 to 0.71], respectively). However, there was a statistically significant difference between blood samples (P < 0.001) and urine samples (P = 0.028). The negative percentage agreements were 97% and 84% and the positive percentage agreements were 68% and 83% for blood and urine samples, respectively. Although the cell culture method is the recommended gold standard, our results show that qPCR assay is a valid alternative method for the rapid and specific detection of pathogenic Leptospira spp. in urine and blood samples during vaccine efficacy studies, without loss of sensitivity.

Evaluation of safety and immunogenicity of feline vaccines with reduced volume.

A non adjuvanted vaccine against feline herpesvirus, feline calicivirus, feline panleucopenia and feline leukemia has been formulated in reduced volume (0.5 ml) with the same antigen content as the conventional 1 ml presentation. This paper reports studies evaluating the safety and the immunogenicity of this reduced volume vaccine in comparison with the conventional volume vaccine. The safety of both vaccines was evaluated in a small sized laboratory trial. It was further tested in a randomized controlled field trial on a total of 398 cats. Immediate and delayed local and systemic adverse events were monitored after vaccination. The immunogenicity of each vaccine was also checked by serological antibody responses against the vaccines antigens during the laboratory trial. These studies showed that the 0.5 ml vaccine was well tolerated in cats, inducing less local events, while keeping the same immunogenicity as the corresponding 1 ml vaccine. Reducing the volume of the vaccine is a way to improve the convenience of administration and to help following vaccination guidelines with the aim of reducing the incidence of adverse events following vaccination.

ß-Glucan as Trained Immunity-Based Adjuvants for Rabies Vaccines in Dogs.

The mechanisms of trained immunity have been extensively described in vitro and the beneficial effects are starting to be deciphered in in vivo settings. Prototypical compounds inducing trained immunity, such as ß-glucans, act through epigenetic reprogramming and metabolic changes of innate immune cells. The recent advances in this field have opened new areas for the development of Trained immunity-based adjuvants (TIbAs). In this study, we assessed in dogs the potential immune training effects of ß-glucans as well as their capacity to enhance the adaptive immune response of an inactivated rabies vaccine (Rabisin®). Injection of ß-glucan from Euglena gracilis was performed 1 month before vaccination with Rabisin® supplemented or not with the same ß-glucan used as adjuvant. Trained innate immunity parameters were assessed during the first month of the trial. The second phase of the study was focused on the ability of ß-glucan to enhance adaptive immune responses measured by multiple immunological parameters. B and T-cell specific responses were monitored to evaluate the immunogenicity of the rabies vaccine adjuvanted with ß-glucan or not. Our preliminary results support that adjuvantation of Rabisin® vaccine with ß-glucan elicit a higher B-lymphocyte immune response, the prevailing factor of protection against rabies. ß-glucan also tend to stimulate the T cell response as shown by the cytokine secretion profile of PBMCs re-stimulated ex vivo. Our data are providing new insights on the impact of trained immunity on the adaptive immune response to vaccines in dogs. The administration of ß-glucan, 1 month before or simultaneously to Rabisin® vaccination give promising results for the generation of new TIbA candidates and their potential to provide increased immunogenicity of specific vaccines.

ß-Glucan-Induced Trained Immunity in Dogs.

Several observations in the world of comparative immunology in plants, insects, fish and eventually mammals lead to the discovery of trained immunity in the early 2010's. The first demonstrations provided evidence that innate immune cells were capable of developing memory after a first encounter with some pathogens. Trained immunity in mammals was initially described in monocytes with the Bacille Calmette-Guerin vaccine (BCG) or prototypical agonists like ß-glucans. This phenomenon relies on epigenetic and metabolic modifications leading to an enhanced secretion of inflammatory cytokines when the host encounters homologous or heterologous pathogens. The objective of our research was to investigate the trained immunity, well-described in mouse and human, in other species of veterinary importance. For this purpose, we adapted an in vitro model of trained innate immunity in dogs. Blood enriched monocytes were stimulated with ß-glucans and we confirmed that it induced an increased production of pro-inflammatory and anti-microbial compounds in response to bacterial stimuli. These results constitute the first demonstration of trained immunity in dogs and confirm its signatures in other mammalian species, with an implication of cellular mechanisms similar to those described in mice and humans regarding cellular epigenetics and metabolic regulations.

Multiple Correspondence Analysis on Amino Acid Properties within the Variable Region of the Capsid Protein Shows Differences between Classical and Virulent Systemic Feline Calicivirus Strains.

Feline calicivirus (FCV) is a widespread and highly prevalent pathogen of domestic cats, responsible for mild upper respiratory tract disease. Outbreaks of severe virulent systemic disease (VSD) associated with FCV infection have been reported worldwide. VSD FCV strains have a broader tropism and cause a systemic vascular compromise. Despite clear differences in the pathogenesis of VSD and oral respiratory infections, attempts to identify specific molecular markers of VSD strains on the major capsid protein VP1 have failed. Region E of VP1 is responsible for the interaction with the cell receptor Junctional Adhesion Molecule JAM-1 (FeJAM-1) and with VP2 minor capsid protein during the entry of the virus. We carried out an original analysis on the sequences from region E of VSD and classical strains. A Multiple Correspondence Analysis was performed on a Boolean matrix built by coding sequences on the basis of their amino acid properties. For the first time, this approach was able to differentiate VSD and classical FCV. Seven remarkable residue positions were shown to be statistically significant for pathotype differentiation, mainly located in the N-terminal hypervariable part of region E. As structural analysis suggested an interaction of these residues with FeJAM-1 or VP2, post-binding events, and specific conformational changes may explain the difference of pathogenesis between pathotypes.

Non-specific Effects of Vaccines Illustrated Through the BCG Example: From Observations to Demonstrations.

Epidemiological studies regarding many successful vaccines suggest that vaccination may lead to a reduction in child mortality and morbidity worldwide, on a grander scale than is attributable to protection against the specific target diseases of these vaccines. These non-specific effects (NSEs) of the Bacille Calmette-Guérin (BCG) vaccine, for instance, implicate adaptive and innate immune mechanisms, with recent evidence suggesting that trained immunity might be a key instrument at play. Collectively referring to the memory-like characteristics of innate immune cells, trained immunity stems from epigenetic reprogramming that these innate immune cells undergo following exposure to a primary stimulus like BCG. The epigenetic changes subsequently regulate cytokine production and cell metabolism and in turn, epigenetic changes are regulated by these effects. Novel -omics technologies, combined with in vitro models for trained immunity and other immunological techniques, identify the biological pathways within innate cells that enable training by BCG. Future research should aim to identify biomarkers for vaccine heterologous effects, such that they can be applied to epidemiological studies. Linking biological mechanisms to the reduction in all-cause mortality observed in epidemiological studies will strengthen the evidence in favor of vaccine NSEs. The universal acceptance of these NSEs would demand a re-evaluation of current vaccination policies, such as the childhood vaccination recommendations by the World Health Organization, in order to produce the maximum impact on childhood mortality.

Development of a highly sensitive PCR/DNA chip method to detect mycoplasmas in a veterinary modified live vaccine.

Mycoplasmas are potential contaminants that introduce undesirable changes in mammalian cell cultures. They frequently contaminate cell substrates and other starting materials used for manufacturing cell-derived biologics, such as vaccines and pharmaceutical products. Mycoplasma purity testing of live vaccines, active ingredients, raw material, and seed lots is required during vaccine production. Previously, testing using a time-consuming, costly 28-day culture assay, which lacks sensitivity for species that do not grow in culture, was required in the European Pharmacopoeia (Ph. Eur). But now nucleic acid amplification techniques (NATs) can be used. NATs provide rapid results and are sensitive. We evaluated the sensitivity and specificity of a commercially-available NAT to detect individual mycoplasma DNA in a veterinary modified live vaccine using five reference strains recommended by the Ph. Eur. Our results showed that this NAT-based method can be used to detect mycoplasma in spiked live vaccine, without interference from the vaccine components, with a limit of detection of 10 CFU/mL, as required by the Ph. Eur. Its specificity was demonstrated since no mycoplasmas were detected in non-spiked vaccine. This method is undergoing validation as a replacement for the conventional culture method in the production of veterinary live vaccines.

Efficacy of a nonadjuvanted recombinant FeLV vaccine and two inactivated FeLV vaccines when subject to consistent virulent FeLV challenge conditions.

The purpose of this study was to compare the efficacy of three FeLV vaccines, under identical conditions in a laboratory challenge model that closely mimics natural infection. Four groups of cats (n = 20 per group) were administered two doses of vaccine, 21 days apart, starting at 9-10 weeks of age (Purevax® FeLV, Versifel® FeLV, Nobivac® feline 2-FeLV, and a placebo). Cats were challenged 3 weeks later with a virulent, heterologous FeLV isolate. FeLV antigenemia was determined at weekly intervals from 3 to 15 weeks postchallenge. Circulating proviral DNA was determined on terminal PBMC samples. Following challenge, persistent antigenemia developed in 15 (75%) placebo-vaccinated cats, 3 (15%) cats in the Versifel FeLV vaccinated group, and 1 cat (5%) each in the Purevax FeLV and the Nobivac FeLV vaccinated groups. The prevented fractions for three vaccine groups were 93%, 93%, and 80% respectively. The adjusted p-values for all vaccine group comparisons fail to approach statistical significance. There was excellent agreement between proviral FeLV DNA in circulating PBMCs and persistent antigenemia. It is shown that when cats are managed under the same conditions during a virulent challenge, via the normal route of infection, the tested vaccines all show a comparable degree of protection.

Multivariate analysis of the immune response to a vaccine as an alternative to the repetition of animal challenge studies for vaccines with demonstrated efficacy.

The assessment of vaccine combinations, or the evaluation of the impact of minor modifications of one component in well-established vaccines, requires animal challenges in the absence of previously validated correlates of protection. As an alternative, we propose conducting a multivariate analysis of the specific immune response to the vaccine. This approach is consistent with the principles of the 3Rs (Refinement, Reduction and Replacement) and avoids repeating efficacy studies based on infectious challenges in vivo. To validate this approach, a set of nine immunological parameters was selected in order to characterize B and T lymphocyte responses against canine rabies virus and to evaluate the compatibility between two canine vaccines, an inactivated rabies vaccine (RABISIN®) and a combined vaccine (EURICAN® DAPPi-Lmulti) injected at two different sites in the same animals. The analysis was focused on the magnitude and quality of the immune response. The multi-dimensional picture given by this 'immune fingerprint' was used to assess the impact of the concomitant injection of the combined vaccine on the immunogenicity of the rabies vaccine. A principal component analysis fully discriminated the control group from the groups vaccinated with RABISIN® alone or RABISIN®+EURICAN® DAPPi-Lmulti and confirmed the compatibility between the rabies vaccines. This study suggests that determining the immune fingerprint, combined with a multivariate statistical analysis, is a promising approach to characterizing the immunogenicity of a vaccine with an established record of efficacy. It may also avoid the need to repeat efficacy studies involving challenge infection in case of minor modifications of the vaccine or for compatibility studies.

A versatile in vitro ELISA test for quantification and quality testing of infectious, inactivated and formulated rabies virus used in veterinary monovalent or combination vaccine.

Regulatory potency test for rabies vaccines requires mice vaccination followed by challenge with a live virus via intracerebral route. An alternative in vitro test, consistent with the "3R's" (Reduce, Replace, Refine) was designed to quantify active glycoprotein G using seroneutralizing monoclonal antibodies. This versatile ELISA targets well conformed neutralizing epitopes. Therefore, it quantifies only the trimeric pre-fusion form of glycoprotein G known to elicits the production of viral neutralizing antibodies. The ELISA makes it possible to quantify the rabies antigen during all steps of the product cycle (i.e. viral cultivation, downstream process, formulation and product stability in the presence of aluminum gel or other vaccine valence). Moreover, the batch-to-batch consistency of our active ingredients and formulated products could be demonstrated.

Three-year duration of immunity for feline herpesvirus and calicivirus evaluated in a controlled vaccination-challenge laboratory trial.

Feline vaccination guidelines recommend less frequent boosters for the core vaccines (rhinotracheitis, calicivirosis and infectious panleucopenia). Most guidelines recommend boosters at 3-yearly intervals after a basic vaccination including primary vaccination and revaccination one year later. The objective of this study was to assess the duration of immunity induced by PUREVAX(®) RCPCh FeLV, a non-adjuvanted vaccine against feline rhinotracheitis, calicivirosis, infectious panleucopenia, chlamydiosis and leukemia. After primary vaccination followed by revaccination one year later with a vaccine formulated at minimum dose, the cats were kept in a confined environment and challenged 3 years later with a virulent heterologous strain of feline calicivirus (FCV) and subsequently a virulent strain of feline herpesvirus (FHV). Clinical signs and viral excretion were recorded for two weeks after each viral inoculation. Contemporary unvaccinated cats and new animals added at the time of challenge were used as controls. The vaccination regimen induced a stable and long-lasting humoral response. Vaccination resulted in a significant reduction in the severity of the disease after FHV challenge and in the frequency of cats showing a severe calicivirosis (defined as a combination of systemic clinical symptoms and oronasal ulcers). As opposed to the significant reduction of excretion observed a few weeks after primo-vaccination or even one year after vaccination for FCV, viral shedding was not reduced 3 years after revaccination. This study showed that primary vaccination and revaccination one year later with PUREVAX(®) RCPCh FeLV was able to induce 3-year duration of immunity against FCV and FHV. The results and conclusion of this study are consistent with current vaccination guidelines and will allow the veterinarian to adapt the vaccination regimen to the way of life of the cat.

Structure determination of feline calicivirus virus-like particles in the context of a pseudo-octahedral arrangement.

The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs) were formed, a majority built of 60 subunits (T=1) and a minority probably built of 180 subunits (T=3). The structure of the small particles was determined by x-ray crystallography at 0.8 nm resolution helped by cryo-electron microscopy in order to understand their formation. Cubic crystals belonged to space group P213. Their self-rotation function showed the presence of an octahedral pseudo-symmetry similar to the one described previously by Agerbandje and co-workers for human parvovirus VLPs. The crystal structure could be solved starting from the published VP1 structure in the context of the T=3 viral capsid. In contrast to viral capsids, where the capsomers are interlocked by the exchange of the N-terminal arm (NTA) domain, this domain is disordered in the T=1 capsid of the VLPs. Furthermore it is prone to proteolytic cleavage. The relative orientation of P (protrusion) and S (shell) domains is alerted so as to fit VP1 to the smaller T=1 particle whereas the intermolecular contacts around 2-fold, 3-fold and 5-fold axes are conserved. By consequence the surface of the VLP is very similar compared to the viral capsid and suggests a similar antigenicity. The knowledge of the structure of the VLPs will help to improve their stability, in respect to a use for vaccination.

A targeted mutation within the feline leukemia virus (FeLV) envelope protein immunosuppressive domain to improve a canarypox virus-vectored FeLV vaccine.

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the "mechanical" function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be "switched off" by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.

Diversity of trends of viremia and T-cell markers in experimental acute feline immunodeficiency virus infection.

OBJECTIVE: The early events of human immunodeficiency virus infection seem critical for progression toward disease and antiretroviral therapy initiation. We wanted to clarify some still unknown prognostic relationships between inoculum size and changes in various immunological and virological markers. Feline immunodeficiency virus infection could be a helpful model. METHODS: Viremia and T-cell markers (number of CD4, CD8, CD8ß(low)CD62L(neg) T-cells, CD4/CD8 ratio, and percentage of CD8ß(low)CD62L(neg) cells among CD8 T-cells) were measured over 12 weeks in 102 cats infected with different feline immunodeficiency virus strains and doses. Viremia and T-cell markers trajectory groups were determined and the dose-response relationships between inoculum titres and trajectory groups investigated. RESULTS: Cats given the same inoculum showed different patterns of changes in viremia and T-cell markers. A statistically significant positive dose-response relationship was observed between inoculum titre and i) viremia trajectory-groups (r = 0.80, p<0.01), ii) CD8ß(low)CD62L(neg) cell-fraction trajectory-groups (r = 0.56, p<0.01). Significant correlations were also found between viremia and the CD4/CD8 ratio and between seven out of ten T-cell markers. CONCLUSIONS: In cats, the infectious dose determines early kinetics of viremia and initial CD8+ T-cell activation. An expansion of the CD8ß(low)CD62L(neg) T-cells might be an early predictor of progression toward disease. The same might be expected in humans but needs confirmation.

True versus false parasite interactions: a robust method to take risk factors into account and its application to feline viruses.

BACKGROUND: Multiple infections are common in natural host populations and interspecific parasite interactions are therefore likely within a host individual. As they may seriously impact the circulation of certain parasites and the emergence and management of infectious diseases, their study is essential. In the field, detecting parasite interactions is rendered difficult by the fact that a large number of co-infected individuals may also be observed when two parasites share common risk factors. To correct for these "false interactions", methods accounting for parasite risk factors must be used. METHODOLOGY/PRINCIPAL FINDINGS: In the present paper we propose such a method for presence-absence data (i.e., serology). Our method enables the calculation of the expected frequencies of single and double infected individuals under the independence hypothesis, before comparing them to the observed ones using the chi-square statistic. The method is termed "the corrected chi-square." Its robustness was compared to a pre-existing method based on logistic regression and the corrected chi-square proved to be much more robust for small sample sizes. Since the logistic regression approach is easier to implement, we propose as a rule of thumb to use the latter when the ratio between the sample size and the number of parameters is above ten. Applied to serological data for four viruses infecting cats, the approach revealed pairwise interactions between the Feline Herpesvirus, Parvovirus and Calicivirus, whereas the infection by FIV, the feline equivalent of HIV, did not modify the risk of infection by any of these viruses. CONCLUSIONS/SIGNIFICANCE: This work therefore points out possible interactions that can be further investigated in experimental conditions and, by providing a user-friendly R program and a tutorial example, offers new opportunities for animal and human epidemiologists to detect interactions of interest in the field, a crucial step in the challenge of multiple infections.