Evaluation of a SARS-CoV-2 spike protein ectodomain subunit vaccine with a squalene emulsion adjuvant in rodents and rhesus macaques.

COVID-19 vaccines have played an important role in reducing the impact of the current pandemic. Previously, we developed NARUVAX-C19 vaccine based on a recombinant Wuhan spike protein extracellular domain expressed in insect cells and formulated with a squalene emulsion adjuvant (Sepivac SWE™). The current study assessed the immunogenicity, efficacy, and safety of NARUVAX-C19 vaccine in rhesus macaques and hamsters. Macaques immunized intramuscularly with two doses of NARUVAX-C19 vaccine showed no adverse effects and demonstrated cellular immunity as assessed by T cell IFN-γ responses against spike protein, in addition to inducing a humoral response. Serum from immunized animals neutralized the homologous wild-type SARS-CoV-2 virus as well as the Alpha and Delta variants. In hamsters, immunization with NARUVAX-C19 vaccine protected against a heterologous challenge with the Delta virus, as reflected by reduced lung and nasal viral loads and lung pathology in immunized animals. Nevertheless, some NARUVAX-C19 vaccinated animals were still shown to transmit infection to naïve sentinel animals. Overall, NARUVAX-C19 vaccine induced broadly cross-neutralizing antibody and T cell IFN-γ responses in rhesus macaques and provided heterologous protection of hamsters against infection by the Delta virus variant. This data supports the utility of squalene emulsion-based adjuvanted recombinant vaccine in protection against SARS-CoV-2 and supports their continued clinical development.

Cytokine response and damages in the lungs of aging Syrian hamsters on a high-fat diet infected with the SARS-CoV-2 virus.

Hypertriglyceridemia, obesity, and aging are among the key risk factors for severe COVID-19 with acute respiratory distress syndrome (ARDS). One of the main prognostic biomarkers of ARDS is the level of cytokines IL-6 and TNF-α in the blood. In our study, we modeled hyperglyceridemia and hypercholesterolemia on 18-month-old Syrian hamsters (Mesocricetus auratus). By 18 months, the animals showed such markers of aging as weight stabilization with a tendency to reduce it, polycystic liver disease, decreased motor activity, and foci of alopecia. The high-fat diet caused an increase in triglycerides and cholesterol, as well as fatty changes in the liver. On the third day after infection with the SARS-CoV-2 virus, animals showed a decrease in weight in the groups with a high-fat diet. In the lungs of males on both diets, there was an increase in the concentration of IFN-α, as well as IL-6 in both males and females, regardless of the type of diet. At the same time, the levels of TNF-α and IFN-γ did not change in infected animals. Morphological studies of the lungs of hamsters with SARS-CoV-2 showed the presence of a pathological process characteristic of ARDS. These included bronchointerstitial pneumonia and diffuse alveolar damages. These observations suggest that in aging hamsters, the immune response to pro-inflammatory cytokines may be delayed to a later period. Hypertriglyceridemia, age, and gender affect the severity of COVID-19. These results will help to understand the pathogenesis of COVID-19 associated with age, gender, and disorders of fat metabolism in humans.

An intranasal vaccine comprising SARS-CoV-2 spike receptor-binding domain protein entrapped in mannose-conjugated chitosan nanoparticle provides protection in hamsters.

We developed a novel intranasal SARS-CoV-2 subunit vaccine called NARUVAX-C19/Nano based on the spike protein receptor-binding domain (RBD) entrapped in mannose-conjugated chitosan nanoparticles (NP). A toll-like receptor 9 agonist, CpG55.2, was also added as an adjuvant to see if this would potentiate the cellular immune response to the NP vaccine. The NP vaccine was assessed for immunogenicity, protective efficacy, and ability to prevent virus transmission from vaccinated animals to naive cage-mates. The results were compared with a RBD protein vaccine mixed with alum adjuvant and administered intramuscularly. BALB/c mice vaccinated twice intranasally with the NP vaccines exhibited secretory IgA and a pronounced Th1-cell response, not seen with the intramuscular alum-adjuvanted RBD vaccine. NP vaccines protected Syrian hamsters against a wild-type SARS-CoV-2 infection challenge as indicated by significant reductions in weight loss, lung viral load and lung pathology. However, despite significantly reduced viral load in the nasal turbinates and oropharyngeal swabs from NP-vaccinated hamsters, virus transmission was not prevented to naïve cage-mates. In conclusion, intranasal RBD-based NP formulations induced mucosal and Th1-cell mediated immune responses in mice and protected Syrian hamsters against SARS-CoV-2 infection but not against viral transmission.

A recombinant Artemisia vulgaris pollen adjuvanted Art v 1 protein-based vaccine treats allergic rhinitis and bronchial asthma using pre- and co-seasonal ultrashort immunotherapy regimens in sensitized mice.

Allergic rhinitis is an important risk factor for bronchial asthma. Allergen-specific immunotherapy (ASIT) is the gold standard for treatment of allergic rhinitis, conjunctivitis, and asthma. A disadvantage of current ASIT methods is the length of therapy which requires numerous allergen administrations. The success of ASIT is determined by its schedule, which, depending on the vaccine and type of allergy, can be pre-seasonal (before the allergy season begins), combined pre/co-seasonal (during the allergy season) etc. The aim of the present study was to evaluate a vaccine based on recombinant Artemisia vulgaris pollen major Art v 1 protein formulated with ISA-51 adjuvant for therapy of allergic rhinitis and bronchial asthma in Artemisia-sensitized mice in an ultrashort (4 subcutaneous injections at weekly intervals) pre- and co-seasonal ASIT regimen. To simulate co-seasonal ASIT in mice, mice were regularly challenged with intranasal and nebulized Artemisia vulgaris pollen extract at the same time as receiving subcutaneous ASIT. For comparison, we used a previous Art v 1 protein vaccine formulated with SWE adjuvant, which in this study was modified by adding CpG oligonucleotide (Th1-biasing synthetic toll-like receptor 9 agonist), and a commercial vaccine containing a modified Artemisia vulgaris extract with aluminum hydroxide adjuvant. The therapeutic potential of Art v 1 based vaccine formulations with different ASIT regimens was evaluated in high and low (10 times lower) dose regimens. The ISA-51-adjuvanted vaccine formulations were the only ones among those studied in the ultrashort pre- and co-seasonal ASIT regimens to provide significant reduction in both signs of allergic rhinitis and bronchial asthma in sensitized mice (vs. positive control). In the ISA-51 adjuvanted group, immune response polarization toward Th1/Treg was observed in pre-seasonal ASIT, as reflected in a significant decrease in the serum level of total and Art v 1-specific IgE and increased ratios of allergen-specific IgG2a/IgG1 and IFN-γ/IL-4. The high dose SWE-CpG-adjuvanted vaccine had similar efficacy to the ISA-51 adjuvanted groups whereas the commercial vaccine showed significantly less effectiveness. The findings support further preclinical safety studies of the Art v 1-based vaccine formulated with ISA-51 adjuvant.

Evaluation of a Novel Adjuvanted Vaccine for Ultrashort Regimen Therapy of Artemisia Pollen-Induced Allergic Bronchial Asthma in a Mouse Model.

Wormwood (Artemisia) pollen is among the top 10 aeroallergens globally that cause allergic rhinitis and bronchial asthma. Allergen-specific immunotherapy (ASIT) is the gold standard for treating patients with allergic rhinitis, conjunctivitis, and asthma. A significant disadvantage of today's ASIT methods is the long duration of therapy and multiplicity of allergen administrations. The goal of this study was to undertake a pilot study in mice of a novel ultrashort vaccine immunotherapy regimen incorporating various adjuvants to assess its ability to treat allergic bronchial asthma caused by wormwood pollen. We evaluated in a mouse model of wormwood pollen allergy candidates comprising recombinant Art v 1 wormwood pollen protein formulated with either newer (Advax, Advax-CpG, ISA-51) or more traditional [aluminum hydroxide, squalene water emulsion (SWE)] adjuvants administered by the intramuscular or subcutaneous route vs. intranasal administration of a mucosal vaccine formulation using chitosan-mannose nanoparticle entrapped with Art v 1 protein. The vaccine formulations were administered to previously wormwood pollen-sensitized animals, four times at weekly intervals. Desensitization was determined by measuring decreases in immunoglobulin E (IgE), cellular immunity, ear swelling test, and pathological changes in the lungs of animals after aeroallergen challenge. Art v 1 protein formulation with Advax, Advax-CpG, SWE, or ISA-51 adjuvants induced a significant decrease in both total and Art v 1-specific IgE with a concurrent increase in Art v 1-specific IgG compared to the positive control group. There was a shift in T-cell cytokine secretion toward a Th1 (Advax-CpG, ISA-51, and Advax) or a balanced Th1/Th2 (SWE) pattern. Protection against lung inflammatory reaction after challenge was seen with ISA-51, Advax, and SWE Art v 1 formulations. Overall, the ISA-51-adjuvanted vaccine group induced the largest reduction of allergic ear swelling and protection against type 2 and non-type 2 lung inflammation in challenged animals. This pilot study shows the potential to develop an ultrashort ASIT regimen for wormwood pollen-induced bronchial asthma using appropriately adjuvanted recombinant Art v 1 protein. The data support further preclinical studies with the ultimate goal of advancing this therapy to human clinical trials.

A Spike Protein-Based Subunit SARS-CoV-2 Vaccine for Pets: Safety, Immunogenicity, and Protective Efficacy in Juvenile Cats.

Whereas, multiple vaccine types have been developed to curb the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) among humans, there are very few vaccines being developed for animals including pets. To combat the threat of human-to-animal, animal-to-animal, and animal-to-human transmission and the generation of new virus variants, we developed a subunit SARS-CoV-2 vaccine which is based on the recombinant spike protein extracellular domain expressed in insect cells and then formulated with appropriate adjuvants. Sixteen 8-12-week-old outbred female and male kittens (n = 4 per group) were randomly assigned into four treatment groups: spike protein alone; spike plus ESSAI oil-in-water (O/W) 1849102 adjuvant; spike plus aluminum hydroxide adjuvant; and a PBS control. All animals were vaccinated intramuscularly twice, 2 weeks apart, with 5 µg of spike protein in a volume of 0.5 ml. On days 0 and 28, serum samples were collected to evaluate anti-spike IgG, antibody inhibition of spike binding to angiotensin-converting enzyme 2 (ACE-2), neutralizing antibodies against wild-type and delta variant viruses, and hematology studies. At day 28, all groups were challenged with SARS-CoV-2 wild-type virus 106 TCID50 intranasally. On day 31, tissue samples (lung, heart, and nasal turbinates) were collected for viral RNA detection, and virus titration. After two immunizations, both vaccines induced high titers of serum anti-spike IgG that inhibited spike ACE-2 binding and neutralized both wild-type and delta variant virus. Both adjuvanted vaccine formulations protected juvenile cats against virus shedding from the upper respiratory tract and viral replication in the lower respiratory tract and hearts. These promising data warrant ongoing evaluation of the vaccine's ability to protect cats against SARS-CoV-2 infection and in particular to prevent transmission.

An adjuvanted subunit SARS-CoV-2 spike protein vaccine provides protection against Covid-19 infection and transmission.

Recombinant protein approaches offer major promise for safe and effective vaccine prevention of SARS-CoV-2 infection. We developed a recombinant spike protein vaccine (called NARUVAX-C19) and characterized its ability when formulated with a nanoemulsion adjuvant to induce anti-spike antibody and T-cell responses and provide protection including against viral transmission in rodent. In mice, NARUVAX-C19 vaccine administered intramuscularly twice at 21-day interval elicited balanced Th1/Th2 humoral and T-cell responses with high titers of neutralizing antibodies against wild-type (D614G) and delta (B.1.617.2) variants. In Syrian hamsters, NARUVAX-C19 provided complete protection against wild-type (D614G) infection and prevented its transmission to naïve animals (n = 2/group) placed in the same cage as challenged animals (n = 6/group). The results contrasted with only weak protection seen with a monomeric spike receptor-binding domain (RBD) vaccine even when formulated with the same adjuvant. These encouraging results warrant the ongoing development of this COVID-19 vaccine candidate.

Building Scientific Capability and Reducing Biological Threats: The Effect of Three Cooperative Bio-Research Programs in Kazakhstan.

Cooperative research programs aimed at reducing biological threats have increased scientific capabilities and capacities in Kazakhstan. The German Federal Foreign Office's German Biosecurity Programme, the United Kingdom's International Biological Security Programme and the United States Defense Threat Reduction Agency's Biological Threat Reduction Program provide funding for partner countries, like Kazakhstan. The mutual goals of the programs are to reduce biological threats and enhance global health security. Our investigation examined these cooperative research programs, summarizing major impacts they have made, as well as common successes and challenges. By mapping various projects across the three programs, research networks are highlighted which demonstrate best communication practices to share results and reinforce conclusions. Our team performed a survey to collect results from Kazakhstani partner scientists on their experiences that help gain insights into enhancing day-to-day approaches to conducting cooperative scientific research. This analysis will serve as a basis for a capability maturity model as used in industry, and in addition builds synergy for future collaborations that will be essential for quality and sustainment.

Operationalizing Cooperative Research for Infectious Disease Surveillance: Lessons Learned and Ways Forward.

The current COVID-19 pandemic demonstrates the need for urgent and on-demand solutions to provide diagnostics, treatment and preventative measures for infectious disease outbreaks. Once solutions are developed, meeting capacities depends on the ability to mitigate technical, logistical and production issues. While it is difficult to predict the next outbreak, augmenting investments in preparedness, such as infectious disease surveillance, is far more effective than mustering last-minute response funds. Bringing research outputs into practice sooner rather than later is part of an agile approach to pivot and deliver solutions. Cooperative multi- country research programs, especially those funded by global biosecurity programs, develop capacity that can be applied to infectious disease surveillance and research that enhances detection, identification, and response to emerging and re-emerging pathogens with epidemic or pandemic potential. Moreover, these programs enhance trust building among partners, which is essential because setting expectation and commitment are required for successful research and training. Measuring research outputs, evaluating outcomes and justifying continual investments are essential but not straightforward. Lessons learned include those related to reducing biological threats and maturing capabilities for national laboratory diagnostics strategy and related health systems. Challenges, such as growing networks, promoting scientific transparency, data and material sharing, sustaining funds and developing research strategies remain to be fully resolved. Here, experiences from several programs highlight successful partnerships that provide ways forward to address the next outbreak.

Significance of High-Containment Biological Laboratories Performing Work During the COVID-19 Pandemic: Biosafety Level-3 and -4 Labs.

High containment biological laboratories (HCBL) are required for work on Risk Group 3 and 4 agents across the spectrum of basic, applied, and translational research. These laboratories include biosafety level (BSL)-3, BSL-4, animal BSL (ABSL)-3, BSL-3-Ag (agriculture livestock), and ABSL-4 laboratories. While SARS-CoV-2 is classified as a Risk Group 3 biological agent, routine diagnostic can be handled at BSL-2. Scenarios involving virus culture, potential exposure to aerosols, divergent high transmissible variants, and zoonosis from laboratory animals require higher BSL-3 measures. Establishing HCBLs especially those at BSL-4 is costly and needs continual investments of resources and funding to sustain labor, equipment, infrastructure, certifications, and operational needs. There are now over 50 BSL-4 laboratories and numerous BSL-3 laboratories worldwide. Besides technical and funding challenges, there are biosecurity and dual-use risks, and local community issues to contend with in order to sustain operations. Here, we describe case histories for distinct HCBLs: representative national centers for diagnostic and reference, nonprofit organizations. Case histories describe capabilities and assess activities during COVID-19 and include capacities, gaps, successes, and summary of lessons learned for future practice.

Development and Evaluation of a Live Attenuated Egg-Based Camelpox Vaccine.

Camelpox is an infectious viral disease of camels reported in all the camel-breeding areas of Africa, north of the equator, the Middle East and Asia. It causes huge economic loss to the camel industry. We developed a live camelpox virus vaccine candidate using an attenuated strain and evaluated its safety, immunogenicity and protective efficacy in camels. The attenuated virus strain was generated from the camelpox wild-type strain M-96 by 40 consecutive passages on the chorioallantoic membrane of 11-day-old embryonated chicken eggs, henceforth called KM-40 strain. Reversion to virulence of the KM-40 strain was evaluated in camels by three serial passages, confirmed its inability to revert to virulence and its overdose administration was also found safe. Studies of immunogenicity and protective efficacy of the candidate vaccine KM-40 strain in camels was carried out using the dose of 5 x 104.0 EID50. Our data showed complete protection against the challenge infection using the virulent wild-type camelpox virus strain M-96 (dose of 105.0 EID50) which was evaluated at 1, 3, 6 and 12 months post vaccination. In summary, our candidate live attenuated egg-based camelpox vaccine strain KM-40 was found safe, protective, and thus has the potential to use safely in field conditions.

Registered Influenza Viral Vector Based Brucella abortus Vaccine for Cattle in Kazakhstan: Age-Wise Safety and Efficacy Studies.

A novel influenza viral vector based Brucella abortus vaccine (Flu-BA) was introduced for use in cattle in Kazakhstan in 2019. In this study, the safety and efficacy of the vaccine was evaluated in male and female cattle at different ages, and during pregnancy as a part of its registration process. Our data demonstrated that the Flu-BA vaccine was safe after prime or booster vaccination in calves (5-7 months old male and female), heifers (15-17 months old) and cows (6-7 years old) and was not abortogenic in pregnant animals. A mild, localized granuloma was observed at the Flu-BA injection site. Vaccinated animals did not show signs of influenza infection or reduced milk production in dairy cows, and the influenza viral vector (IVV) was not recovered from nasal swabs or milk. Vaccinated animals in all age groups demonstrated increased IgG antibody responses against Brucella Omp16 and L7/L12 proteins with calves demonstrating the greatest increase in humoral responses. Following experimental challenge with B. abortus 544, vaccinates demonstrated greater protection and no signs of clinical disease, including abortion, were observed. The vaccine effectiveness against B. abortus 544 infection was 75, 60 and 60%, respectively, in calves, heifers and adult cows. Brucella were not isolated from calves of vaccinated cattle that were experimentally challenged during pregnancy. Our data suggests that the Flu-BA vaccine is safe and efficacious in cattle, including pregnant animals; and can therefore be administered to cattle of any age.

Cooperative Research and Infectious Disease Surveillance: A 2021 Epilogue.

As the world looks forward to turning a corner in the face of the COVID-19 pandemic, it becomes increasingly evident that international research cooperation and dialogue is necessary to end this global catastrophe. Last year, we initiated a research topic: "Infectious Disease Surveillance: Cooperative Research in Response to Recent Outbreaks, Including COVID-19," which aimed at featuring manuscripts focused on the essential link between surveillance and cooperative research for emerging and endemic diseases, and highlighting scientific partnerships in countries under-represented in the scientific literature. Here we recognize the body of work published from our manuscript call that resulted in over 50 published papers. This current analysis describes articles and authors from a variety of funded and unfunded international sources. The work exemplifies successful research and publications which are frequently cooperative, and may serve as a basis to model further global scientific engagements.

A Case History in Cooperative Biological Research: Compendium of Studies and Program Analyses in Kazakhstan.

Kazakhstan and the United States have partnered since 2003 to counter the proliferation of weapons of mass destruction. The US Department of Defense (US DoD) has funded threat reduction programs to eliminate biological weapons, secure material in repositories that could be targeted for theft, and enhance surveillance systems to monitor infectious disease outbreaks that would affect national security. The cooperative biological research (CBR) program of the US DoD's Biological Threat Reduction Program has provided financing, mentorship, infrastructure, and biologic research support to Kazakhstani scientists and research institutes since 2005. The objective of this paper is to provide a historical perspective for the CBR involvement in Kazakhstan, including project chronology, successes and challenges to allow lessons learned to be applied to future CBR endeavors. A project compendium from open source data and interviews with partner country Kazakhstani participants, project collaborators, and stakeholders was developed utilizing studies from 2004 to the present. An earlier project map was used as a basis to determine project linkages and continuations during the evolution of the CBR program. It was determined that consistent and effective networking increases the chances to collaborate especially for competitive funding opportunities. Overall, the CBR program has increased scientific capabilities in Kazakhstan while reducing their risk of biological threats. However, there is still need for increased scientific transparency and an overall strategy to develop a capability-based model to better enhance and sustain future research. Finally, we offer a living perspective that can be applied to further link related studies especially those related to One Health and zoonoses and the assessment of similar capability-building programs.

Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs.

Swine influenza virus (SwIV) is one of the important zoonotic pathogens. Current flu vaccines have failed to provide cross-protection against evolving viruses in the field. Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable FDA approved polymer and widely used in drug and vaccine delivery. In this study, inactivated SwIV H1N2 antigens (KAg) encapsulated in PLGA nanoparticles (PLGA-KAg) were prepared, which were spherical in shape with 200 to 300nm diameter, and induced maturation of antigen presenting cells in vitro. Pigs vaccinated twice with PLGA-KAg via intranasal route showed increased antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/memory and cytotoxic T cells (CTLs) in peripheral blood mononuclear cells (PBMCs). In PLGA-KAg vaccinated and heterologous SwIV H1N1 challenged pigs, clinical flu symptoms were absent, while the control pigs had fever for four days. Grossly and microscopically, reduced lung pathology and viral antigenic mass in the lung sections with clearance of infectious challenge virus in most of the PLGA-KAg vaccinated pig lung airways were observed. Immunologically, PLGA-KAg vaccine irrespective of not significantly boosting the mucosal antibody response, it augmented the frequency of IFN-γ secreting total T cells, T-helper and CTLs against both H1N2 and H1N1 SwIV. In summary, inactivated influenza virus delivered through PLGA-NPs reduced the clinical disease and induced cross-protective cell-mediated immune response in a pig model. Our data confirmed the utility of a pig model for intranasal particulate flu vaccine delivery platform to control flu in humans.

First evaluation of an influenza viral vector based Brucella abortus vaccine in sheep and goats: Assessment of safety, immunogenicity and protective efficacy against Brucella melitensis infection.

Previously we developed and evaluated a candidate influenza viral vector based Brucella abortus vaccine (Flu-BA) administered with a potent adjuvant Montanide Gel01 in cattle, which was found safe and highly effective. This study was aimed to establish a proof-of-concept of the efficacy of Flu-BA vaccine formulation in sheep and goats. We vaccinated sheep and goats with Flu-BA vaccine and as a positive control vaccinated a group of animals with a commercial B. melitensis Rev.1 vaccine. Clinically, both Flu-BA and Rev.1 vaccines were found safe. Serological analysis showed the animals received Flu-BA vaccine did not induce antibody response against Brucella Omp16 and L7/L12 proteins during the period of our study (56days post-initial vaccination, PIV). But observed significant antigen-specific T cell response indicated by increased lymphocyte stimulation index and enhanced secretion of IFN-γ at day 56 PIV in Flu-BA group. The Flu-BA vaccinated animals completely protected 57.1% of sheep and 42.9% of goats against B. melitensis 16M challenge. The severity of brucellosis in terms of infection index and colonization of Brucella in tissues was significantly lower in the Flu-BA group compared to negative control animals group. Nevertheless, positive control commercial Rev.1 vaccine provided strong antigen-specific T cell immunity and protection against B. melitensis 16M infection. We conclude that the Flu-BA vaccine induces a significant antigen-specific T-cell response and provides complete protection in approximately 50% of sheep and goats against B. melitensis 16M infection. Further investigations are needed to improve the efficacy of Flu-BA and explore its practical application in small ruminants.

Inactivated porcine reproductive and respiratory syndrome virus vaccine adjuvanted with Montanide™ Gel 01 ST elicits virus-specific cross-protective inter-genotypic response in piglets.

The efficacy of a novel BEI-inactivated porcine reproductive and respiratory syndrome virus (PRRSV) candidate vaccine in pigs, developed at RIBSP Republic of Kazakhstan and delivered with an adjuvant Montanide™ Gel 01 ST (D/KV/ADJ) was compared with a commercial killed PRRSV vaccine (NVDC-JXA1, C/KV/ADJ) used widely in swine herds of the Republic of Kazakhstan. Clinical parameters (body temperature and respiratory disease scores), virological and immunological profiles [ELISA and virus neutralizing (VN) antibody titers], macroscopic lung lesions and viral load in the lungs (quantitative real-time PCR and cell culture assay) were assessed in vaccinated and both genotype 1 and 2 PRRSV challenged pigs. Our results showed that the commercial vaccine failed to protect pigs adequately against the clinical disease, viremia and lung lesions caused by the challenged field isolates, Kazakh strains of PRRSV type 1 and type 2 genotypes. In contrast, clinical protection, absence of viremia and lung lesions in D/KV/ADJ vaccinated pigs was associated with generation of VN antibodies in both homologous vaccine strain LKZ/2010 (PRRSV type 2) and a heterogeneous type 1 PRRSV strain (CM/08) challenged pigs. Thus, our data indicated the induction of cross-protective VN antibodies by D/KV/ADJ vaccine, and importantly demonstrated that an inactivated PRRSV vaccine could also induce cross-protective response across the viral genotype.

The pathogenicity of swan derived H5N1 virus in birds and mammals and its gene analysis.

BACKGROUND: Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to circulate in poultry and can infect and cause mortality in birds and mammals; the genetic determinants of their increased virulence are largely unknown. The main purpose of this work was to determine the correlation between known molecular determinants of virulence in different avian influenza virus (AIV) genes and the results of experimental infection of birds and mammals with AIV strain A/swan/Mangistau/3/06 (H5N1; SW/3/06). METHODS AND RESULTS: We examined the virulence of SW/3/06 in four species of birds (chickens, ducks, turkeys, geese) and five species of mammals (mice, guinea pigs, cats, dogs, pigs), and identified the molecular determinants of virulence in 11 genes (HA, NA, PB1, PB1-F2, PB2, PA, NS1, NS2, M1, M2 and NP). SW/3/06 does not possess the prime virulence determinant of HPAIV - a polybasic HA cleavage site - and is highly pathogenic in chickens. SW/3/06 replicated efficiently in chickens, ducks, turkeys, mice and dogs, causing 100% mortality within 1.6-5.2 days. In addition, no mortalities were observed in geese, guinea pigs, cats and pigs. The HI assay demonstrated all not diseased animals infected with the SW/3/06 virus had undergone seroconversion by 14, 21 and 28 dpi. Eleven mutations in the seven genes were present in SW/3/06. These mutations may play a role in the pathogenicity of this strain in chickens, ducks, turkeys, mice and dogs. Together or separately, mutations 228S-103S-318I in HA may play a role in the efficient replication of SW/3/06 in mammals (mice, dogs, pigs). CONCLUSIONS: This study provides new information on the pathogenicity of the newly-isolated swan derived H5N1 virus in birds and mammals, and explored the role of molecular determinants of virulence in different genes; such studies may help to identify key virulence or adaptation markers that can be used for global surveillance of viruses threatening to emerge into the human population.