Recombinant Bovine parainfluenza 3 virus Hemagglutinin-neuraminidase (HN)

What is the structural and functional significance of Bovine Parainfluenza 3 Virus Hemagglutinin-neuraminidase?

Bovine Parainfluenza 3 Virus Hemagglutinin-neuraminidase (BPIV3 HN) is a type II surface glycoprotein that serves multiple essential functions in the viral life cycle. Structurally, it is a transmembrane protein that extends from the viral envelope and contains both receptor-binding and enzymatic domains. The HN protein plays crucial roles in receptor binding, membrane penetration, syncytium formation, and activation of the fusion (F) protein to initiate fusion—essential processes for viral entry into host cells .

The functional significance of HN lies in its dual activities: hemagglutinin activity that mediates attachment to sialic acid receptors on host cells, and neuraminidase activity that facilitates viral release by cleaving sialic acid residues. Studies indicate that similar to human parainfluenza virus HN proteins, BPIV3 HN likely recognizes both α2,3- and α2,6-linked sialic acids, which are important for viral attachment and entry . This dual functionality makes HN a key determinant of viral tropism and an important target for antiviral strategies.

How does recombinant BPIV3 HN protein differ from native viral HN protein?

Recombinant BPIV3 HN protein production typically involves isolating the target gene, inserting it into an expression vector (often with tags for purification purposes), and expressing it in systems such as E. coli, insect cells, or mammalian cells . The key differences from native viral HN include:

These differences can affect immunogenicity, enzymatic activity, and receptor binding properties. When designing experiments, researchers must consider that recombinant HN proteins produced in bacteria (like E. coli) lack post-translational modifications, particularly glycosylation, which can significantly impact protein folding and function . For applications requiring fully functional HN, mammalian or insect cell expression systems are often preferable despite their higher cost and complexity.

What roles do BPIV3 F and HN glycoproteins play in viral pathogenesis and host immune response?

The F and HN glycoproteins of BPIV3 serve complementary roles in viral pathogenesis and subsequently in host immune responses:

In viral pathogenesis:

• HN protein mediates initial attachment to host cells through binding to sialic acid-containing receptors

• HN activates the F protein, which triggers the fusion of viral and cellular membranes

• Together, they facilitate viral entry, spread via cell-cell fusion, and eventual release of new virions

• Both proteins contribute to the host range restriction and attenuation phenotype of BPIV3 in primates

In host immune response:

• Both F and HN glycoproteins are primary targets for neutralizing antibodies

• Co-expression of F and HN proteins has been shown to stimulate higher antibody titers than either protein alone

• Immunization with recombinant vectors expressing both proteins induces significantly higher levels of IgG, neutralizing antibodies (NAb), and hemagglutination inhibition (HI) antibodies

• The proteins also stimulate cellular immunity, with studies showing increased percentages of CD3+/CD8+ T lymphocytes and IL-4+ cytokines in vaccinated animals

Research demonstrates that these glycoproteins, particularly when expressed together, are major contributors to protective immunity against BPIV3 and related viruses .

How do chimeric recombinants containing BPIV3 and HPIV3 F and HN genes affect viral replication and immunogenicity?

Chimeric recombinants containing BPIV3 and HPIV3 F and HN genes demonstrate complex effects on viral replication and immunogenicity, providing valuable insights for vaccine development. Studies with recombinant HPIV3 containing BPIV3 F and HN genes (and the reciprocal construct) have revealed several key findings:

Replication characteristics:

Immunogenicity profile:

• Despite restricted replication in the respiratory tract of rhesus monkeys, rBPIV3-F HHN H (BPIV3 backbone with HPIV3 F and HN genes) conferred protection against HPIV3 challenge that was comparable to that induced by wild-type HPIV3

• The chimeric viruses maintain the antigenic specificity of the virus contributing the F and HN genes while acquiring the attenuation phenotype associated with the viral backbone

These findings have significant implications for vaccine development, as they demonstrate the possibility of creating attenuated chimeric viruses that maintain protective immunogenicity while exhibiting restricted replication in the host.

What are the comparative advantages of different expression systems for producing recombinant BPIV3 HN protein?

Different expression systems offer distinct advantages and limitations for producing recombinant BPIV3 HN protein:

For functional studies of BPIV3 HN, insect cell systems have demonstrated success, as evidenced by the development of a subunit vaccine (HN-Baculo) incorporating the BPIV3 HN protein using a baculovirus expression system . This system produced HN protein that induced elevated levels of specific neutralizing antibody serum and HI antibodies in calves .

The choice of expression system should be guided by the specific research application. For example, if studying receptor binding or developing a vaccine candidate, a system that provides proper glycosylation (insect or mammalian cells) would be essential for maintaining the protein's functional properties.

How does co-expression of F and HN proteins enhance immunogenicity compared to individual protein expression?

Co-expression of F and HN proteins has been demonstrated to significantly enhance immunogenicity compared to individual protein expression through several synergistic mechanisms:

Enhanced antibody responses:

• Recombinant adenovirus expressing both F and HN glycoproteins (rHAd5-F+HN) induced significantly higher titers of IgG antibodies in mice compared to adenoviruses expressing either F (rHAd5-F) or HN (rHAd5-HN) alone

• After boosting with rHAd5-F+HN, mice produced antibody levels against BPIV3 genotypes A and C that exceeded those produced by individual protein expression

• The co-expression approach resulted in higher titers of neutralizing antibodies (NAb) and hemagglutination inhibition (HI) antibodies

Improved cellular immunity:

• Co-expression led to higher percentages of splenic CD3+/CD8+ T lymphocytes and increased IL-4+ cytokines compared to control groups

• This enhanced cellular immune response is critical for defense against respiratory viruses

Protective efficacy:

• Mice immunized with rHAd5-F+HN exhibited much lower viral loads in the lungs and tracheas compared to control groups

• The lungs of mice vaccinated with rHAd5-F+HN showed no notable histopathological changes after challenge, indicating superior protection

The potential mechanisms behind this enhanced effect may include:

• Conformational influence - one protein may influence the conformation of the other, similar to observations with RSV where the G protein influenced F protein conformation

• Complementary immune stimulation - F and HN proteins may stimulate different components of the immune system that work synergistically

• Mimicking of natural virus structure - co-expression better resembles the natural presentation of these antigens on viral particles

In calves, following booster immunization with rHAd5-F+HN, the serum antibody levels against BPIV3 genotype C strain reached impressive levels: 1:20,452 (ELISA), 1:1024 (HI), and 1:426 (NAb) .

What are the optimal protocols for evaluating recombinant BPIV3 HN immunogenicity in animal models?

Evaluating the immunogenicity of recombinant BPIV3 HN requires comprehensive protocols addressing both humoral and cellular immune responses in appropriate animal models:

Animal model selection:

• BALB/c mice are widely acknowledged as an optimal initial animal model for studying respiratory tract infections caused by BPIV3

• For more translational research, calves represent the natural host for BPIV3 and should be included in advanced testing

• Rhesus monkeys and chimpanzees serve as important models for evaluating cross-species protection when developing vaccines against human parainfluenza viruses

Immunization protocol:

• Prime-boost strategy is recommended, with 2-4 weeks between immunizations

• For adenoviral vectors, intramuscular injection is commonly used at doses of 1×10^8 - 1×10^9 PFU for mice and 1×10^9 - 1×10^10 PFU for calves

• Include appropriate control groups: vector-only, irrelevant antigen, and non-immunized controls

Humoral immunity assessment:

• Enzyme-linked immunosorbent assay (ELISA) to measure total IgG and IgG subclasses specific to BPIV3 HN

• Hemagglutination inhibition (HI) assay to evaluate functional antibodies that can block hemagglutination activity

• Virus neutralization assay to determine neutralizing antibody (NAb) titers against both homologous and heterologous BPIV3 genotypes

Cellular immunity assessment:

• Flow cytometry to analyze lymphocyte populations, particularly CD3+/CD8+ T cells

• Cytokine profiling (ELISPOT or intracellular cytokine staining) focusing on IL-4, IFN-γ, and other relevant cytokines

• Lymphocyte proliferation assay upon re-stimulation with viral antigens

Challenge studies:

• Challenge with virulent BPIV3 (typically 14-28 days after final immunization)

• Viral load determination in respiratory tissues using quantitative PCR or plaque assays

• Histopathological assessment of lung and tracheal tissues

• Clinical scoring of respiratory symptoms

Following this comprehensive approach provides robust evaluation of both protective efficacy and the immunological mechanisms involved in response to recombinant BPIV3 HN protein vaccines.

What molecular techniques are most effective for generating recombinant chimeric parainfluenza viruses expressing heterologous antigens?

Generating recombinant chimeric parainfluenza viruses expressing heterologous antigens requires sophisticated molecular techniques. Based on the available literature, the most effective approaches include:

Reverse genetics system:

• The foundation for generating recombinant parainfluenza viruses is a reliable reverse genetics system

• This typically involves a cDNA clone of the full viral genome under control of a T7 or RNA polymerase I promoter

• Support plasmids expressing viral polymerase components (N, P, and L proteins) are co-transfected

• The system allows for precise genetic manipulation and recovery of infectious recombinant viruses

Insertion strategies for heterologous antigens:

• Additional transcription unit (ATU) approach:

  • Introduction of a new gene at intergenic regions under control of PIV3 transcription signals

  • Positioning of the heterologous gene affects expression level (promoter-proximal position generally yields higher expression)

  • Example: RSV G and F open reading frames (ORFs) placed under PIV3 transcription signals and inserted into the rB/HPIV3 genome in the promoter-proximal position

• Chimeric glycoprotein approach:

  • Replacement of PIV3 F and/or HN genes with counterparts from related viruses

  • Creates chimeric viruses with altered host range and antigenic properties

  • Example: rB/HPIV3, a recombinant BPIV3 in which the F and HN genes were replaced with HPIV3 counterparts

• Co-expression strategies:

  • Use of 2A peptide sequences to enable co-expression of multiple proteins from a single transcript

  • Example: rHAd5-F+HN using P2A peptide to enable co-expression of the F and HN proteins

  • Western blot analysis confirmed complete cleavage and strong biological activity of both proteins

Considerations for optimal expression:

• Gene insertion can affect viral replication and must be carefully positioned

• Foreign gene size constraints exist (typically <5 kb for stable maintenance)

• Codon optimization for the viral backbone may improve expression

• Inclusion of appropriate signal sequences for glycoprotein processing and trafficking

These molecular approaches have successfully produced recombinants like rB/HPIV3 expressing RSV glycoproteins for creating bivalent mucosal vaccines against RSV and HPIV3 , and chimeric viruses containing BPIV3 F and HN glycoprotein genes in HPIV3 backbone for studying host restriction .

How can researchers effectively measure cross-neutralization between BPIV3 strains and related parainfluenza viruses?

Measuring cross-neutralization between BPIV3 strains and related parainfluenza viruses is critical for understanding protective immunity and developing broadly protective vaccines. Based on current research methodologies, the following approaches are most effective:

Standardized virus neutralization assay:

• Plaque reduction neutralization test (PRNT):

  • Serial dilutions of test sera are incubated with a fixed amount of virus

  • The mixture is added to susceptible cells and incubated

  • Plaques are counted and compared to control wells

  • Calculate PRNT50 or PRNT80 (serum dilution reducing plaques by 50% or 80%)

  • This method works well for comparing neutralization across genotypes

• Microneutralization assay:

  • Similar to PRNT but performed in microtiter plates

  • Viral cytopathic effect (CPE) is scored or quantified by viability assays

  • More suited for high-throughput screening of multiple sera or virus strains

Test panel design:

• Include representatives of all BPIV3 genotypes (A, B, and C)

• Include related human parainfluenza viruses for cross-species neutralization assessment

• Use well-characterized reference strains alongside contemporary field isolates

• Standardize virus input (typically 100 TCID50 or PFU)

Data analysis and interpretation:

• Calculate neutralizing antibody titers for each virus-serum combination

• Generate cross-neutralization tables showing the fold-difference in neutralization between homologous and heterologous strains

• Apply antigenic cartography to visualize relationships between strains

Studies have shown that recombinant adenovirus rHAd5-F+HN induced cross-neutralizing antibodies in mice against different genotypes of BPIV3, with particularly strong titers recorded against the homologous genotype C strain . Similarly, chimeric virus rBPIV3-F HHN H conferred protection against HPIV3 challenge that was comparable to that induced by wild-type HPIV3 , demonstrating significant cross-protection potential.

How should researchers interpret apparent contradictions in immune response data between different animal models?

Interpreting contradictions in immune response data between different animal models requires a systematic approach that considers multiple factors affecting immune responses to recombinant BPIV3 HN:

Sources of inter-model variability:

• Species-specific differences in respiratory tract physiology:

  • BALB/c mice have significant anatomical and physiological differences compared to bovines and primates

  • Mice are not natural hosts for BPIV3, which affects viral replication and immune responses

  • Receptor distribution and density in the respiratory tract vary across species

• Pre-existing immunity effects:

  • Natural hosts may have pre-existing immunity to related pathogens

  • Calves may have maternal antibodies that interfere with vaccine responses

  • Previous exposures to adenovirus vectors can significantly affect immune efficacy

• Immunological maturity:

  • Laboratory mice typically have naive immune systems

  • Calves and other natural hosts may have more developed immune systems due to environmental exposures

Methodological approach to resolving contradictions:

• Comparative immunology analysis:

  • Examine both quantitative (antibody titers) and qualitative (antibody functions) aspects

  • Compare responses to the same antigen across species using standardized assays

  • Identify conserved versus species-specific immune response patterns

• Dose-response relationships:

  • Differences may simply reflect non-linear dose-response relationships

  • Construct dose-response curves for each animal model

  • Normalize responses based on body weight or respiratory tract surface area

• Temporal dynamics consideration:

  • Immune kinetics differ between species

  • Sample at multiple timepoints to construct response curves

  • Compare peak responses rather than responses at identical timepoints

Integration framework:

When faced with contradictory data, researchers should apply a weighted evidence approach that considers:

• Relevance of each model to the target species (higher weight to natural host)

• Consistency of findings across independent studies

• Biological plausibility of observed differences

• Correlation with protection in challenge studies

For example, in studies with rHAd5-F+HN, while mice showed strong cross-genotype protection, calves exhibited variable responses with higher titers against homologous genotype C (1:1024 HI, 1:426 NAb) compared to genotype A (1:213 HI, 1:85 NAb) . This pattern likely reflects evolutionary differences between BPIV3 genotypes and species-specific immune recognition patterns rather than contradicting the vaccine's efficacy.

What statistical approaches are most appropriate for analyzing neutralizing antibody data from recombinant BPIV3 HN vaccination studies?

Analyzing neutralizing antibody data from recombinant BPIV3 HN vaccination studies requires specialized statistical approaches to account for the unique characteristics of serological data:

Data transformation and normalization:

• Neutralizing antibody titers typically follow a log-normal distribution

• Log2 or log10 transformation of antibody titers is recommended before statistical analysis

• For data sets including zero values (undetectable titers), add a small constant (e.g., 0.5) before transformation

• Consider normalizing to pre-vaccination titers when evaluating responses in animals with pre-existing immunity

Appropriate statistical tests:

• For comparing two groups:

  • Paired t-test for pre- vs. post-vaccination within the same animals

  • Mann-Whitney U test for non-normally distributed data

  • Welch's t-test when variances differ significantly between groups

• For multiple group comparisons:

  • One-way ANOVA with appropriate post-hoc tests (Tukey's HSD, Dunnett's test for comparison to control)

  • Kruskal-Wallis with Dunn's post-hoc test for non-parametric analysis

  • Mixed effects models for repeated measures with multiple variables

• For correlation analysis:

  • Spearman's rank correlation for relating antibody titers to other immune parameters

  • Linear regression on log-transformed data to quantify relationships

Advanced analytical approaches:

• Responder analysis:

  • Define threshold for "seroconversion" (e.g., 4-fold increase in titer)

  • Calculate response rates and compare using chi-square or Fisher's exact test

  • Useful for evaluating protection potential in heterogeneous populations

• Antigenic cartography:

  • Dimensional reduction techniques to map antigenic relationships

  • Useful for visualizing cross-neutralization between BPIV3 genotypes

  • Helps identify antigenic clusters and escape mutants

• Mixed-effects modeling:

  • Accounts for individual variation and repeated measures

  • Can incorporate fixed effects (vaccine type, dose, route) and random effects (individual, litter)

  • Particularly valuable for longitudinal studies tracking antibody persistence

Reporting standards:

In practical application, studies evaluating rHAd5-F+HN have employed these approaches to demonstrate statistically significant differences in antibody responses between vaccination groups, showing that the recombinant expressing both F and HN induced significantly higher titers than constructs expressing single proteins .

How can researchers determine the minimal protective titer of antibodies against recombinant BPIV3 HN in different host species?

Determining the minimal protective titer of antibodies against recombinant BPIV3 HN across different host species is a complex but crucial aspect of vaccine development. The following methodological approach outlines a comprehensive strategy:

Challenge study design:

• Dose-ranging immunization:

  • Administer varying doses of the recombinant HN vaccine

  • Create a spectrum of antibody responses in the test population

  • Include positive controls (natural infection or established vaccine) and negative controls

• Standardized challenge model:

  • Use a well-characterized challenge strain (preferably homologous to vaccine)

  • Standardize challenge dose and route across studies

  • Define clear clinical and virological endpoints (viral shedding, clinical scores, lung pathology)

• Comprehensive sampling:

  • Collect pre-challenge sera for antibody measurements

  • Monitor viral loads in appropriate respiratory specimens post-challenge

  • Document clinical parameters using standardized scoring systems

Statistical determination of protective thresholds:

• Receiver Operating Characteristic (ROC) curve analysis:

  • Plot sensitivity versus 1-specificity for various antibody titer cutpoints

  • Define "protection" based on appropriate endpoint (e.g., absence of viral shedding)

  • Determine optimal cutpoint that maximizes both sensitivity and specificity

  • Calculate area under curve (AUC) to assess the predictive value of antibody titers

• Logistic regression modeling:

  • Model probability of protection as function of log-transformed antibody titers

  • Calculate ED50 (titer providing 50% protection) and ED90 values

  • Incorporate additional variables (age, species, challenge dose) into multivariate models

• Survival analysis approaches:

  • Use time-to-event analysis for outcomes like time to cessation of viral shedding

  • Apply Cox proportional hazards models with antibody titer as predictor

  • Generate hazard ratios associated with specific increases in antibody titers

Cross-species considerations:

Studies should account for species-specific factors:

• Differences in receptor distribution and density

• Variation in innate immune responses

• Species-specific correlates of protection (NAb vs. HI vs. ELISA titers)

Based on available research, protective thresholds appear to differ between species:

• In mice, rHAd5-F+HN vaccination resulting in HI titers of approximately 1:64-1:128 provided protection against BPIV3 challenge

• In calves, NAb titers of 1:85 against heterologous strains showed partial protection, while titers of 1:426 against homologous strains provided more robust protection

• In rhesus monkeys, rBPIV3-F HHN H conferred protection against HPIV3 challenge despite restricted replication, suggesting that even modest local antibody responses may be protective in certain contexts

The minimal protective titer likely varies based on the specific endpoint being assessed (sterilizing immunity vs. prevention of severe disease), highlighting the importance of defining clear protection criteria in study designs.

What are the key unresolved questions regarding the role of BPIV3 HN in viral pathogenesis and immunity?

Despite significant advances in understanding BPIV3 HN, several crucial knowledge gaps remain that deserve research attention:

Structural-functional relationships:

• The precise structural elements of BPIV3 HN that determine host range restriction remain incompletely characterized

• How specific amino acid residues in HN influence receptor binding specificity across different host species requires further elucidation

• The mechanisms by which HN triggers the conformational changes in F protein remain poorly understood at the molecular level

• The dynamics of HN-receptor interactions during different phases of infection need better characterization

Host-pathogen interactions:

• The exact contribution of HN to immune evasion strategies is not fully defined

• How HN-specific antibodies interfere with different functional domains (receptor binding vs. neuraminidase activity) remains unclear

• The extent to which glycosylation patterns affect HN antigenicity and immune recognition across species boundaries requires further study

• The role of HN in modulating innate immune responses in the respiratory mucosa needs clarification

Viral evolution and cross-protection:

• The immunological consequences of genetic drift in HN across BPIV3 genotypes are not completely understood

• How sequence variations in HN across different genotypes affect cross-neutralization remains partially characterized

• The evolutionary constraints on HN that maintain functionality while allowing immune escape need further investigation

• The mechanisms underlying cross-protection between bovine and human parainfluenza viruses require more detailed analysis

Developmental research priorities:

• Comparative structural studies of HN proteins from different BPIV3 genotypes and related paramyxoviruses

• Comprehensive mapping of B and T cell epitopes on HN across species

• Systems biology approaches to understand the impact of HN on host immune signaling pathways

• Development of improved animal models that better recapitulate natural infection patterns

Addressing these knowledge gaps would significantly advance our understanding of BPIV3 HN biology and improve rational vaccine design strategies. The finding that BPIV3 sequences outside the F and HN region also contribute to attenuation in primates emphasizes the importance of studying HN in the context of the complete viral genome.

What novel approaches could enhance the immunogenicity and cross-protection of recombinant BPIV3 HN-based vaccines?

Several innovative strategies show promise for enhancing the immunogenicity and cross-protection of recombinant BPIV3 HN-based vaccines:

Structural design innovations:

• Consensus sequence approaches:

  • Engineer HN antigens based on consensus sequences across multiple genotypes

  • Target conserved epitopes while minimizing strain-specific regions

  • Bioinformatic analysis of epitope conservation across genotypes can guide design

• Structure-guided antigen modifications:

  • Stabilize HN in prefusion conformations to present neutralizing epitopes optimally

  • Remove or mask immunodominant but non-neutralizing epitopes

  • Create chimeric HN proteins incorporating protective epitopes from multiple genotypes

• Multi-component designs:

  • Co-express F and HN proteins to enhance immunogenicity through synergistic effects

  • Incorporate additional viral antigens to broaden protection

  • Design polyvalent formulations covering multiple BPIV3 genotypes

Delivery platform innovations:

• Advanced vector systems:

  • Explore next-generation adenoviral vectors with reduced pre-existing immunity issues

  • Investigate mRNA delivery of BPIV3 HN for direct in vivo expression

  • Develop replication-competent but attenuated viral vectors for mucosal delivery

• Nanoparticle and VLP approaches:

  • Present HN in multimeric arrays on nanoparticles to enhance B cell activation

  • Create virus-like particles (VLPs) displaying both F and HN in their natural configuration

  • Incorporate adjuvant molecules into nanoparticle designs for enhanced immunogenicity

• Prime-boost strategies:

  • Heterologous prime-boost regimens using different delivery platforms

  • Mucosal priming followed by systemic boosting to enhance both local and systemic immunity

  • DNA prime-protein boost approaches to enhance both humoral and cellular responses

Immunomodulatory approaches:

• Targeted adjuvants:

  • TLR agonists tailored to specific immune pathways relevant for respiratory protection

  • Mucosal adjuvants to enhance local IgA production

  • Cytokine-adjuvanted formulations to direct specific immune profiles

• Immune focusing strategies:

  • Glycan modification to direct immune responses to specific epitopes

  • Epitope scaffolding to present conserved neutralizing determinants

  • Masking of immunodominant variable epitopes

Based on current research, the co-expression of F and HN proteins using strategies like the P2A peptide approach has already demonstrated superior immunogenicity compared to single protein expression . Further enhancing this approach with optimized delivery systems and adjuvants could significantly improve vaccine performance across diverse host species and against different viral genotypes.

How might recombinant BPIV3 HN technology be applied to develop multivalent vaccines against related respiratory pathogens?

Recombinant BPIV3 HN technology offers significant potential for developing multivalent vaccines targeting multiple respiratory pathogens:

Vector-based multivalent approaches:

• Chimeric paramyxovirus vectors:

  • Utilize rB/HPIV3 (recombinant BPIV3 with HPIV3 F and HN genes) as a backbone for expressing additional antigens

  • Insert foreign genes at intergenic regions under control of PIV3 transcription signals

  • The natural tropism for respiratory epithelium makes these vectors ideal for respiratory pathogen vaccination

  • Example: rB/HPIV3 expressing RSV G and F open reading frames creates a bivalent vaccine against HPIV3 and RSV

• Adenovirus vector platforms:

  • Building on the rHAd5-F+HN model, incorporate additional respiratory pathogen antigens

  • Use of 2A peptides allows co-expression of multiple antigens from a single transcript

  • Human adenovirus vectors show minimal cross-reactivity with bovine adenoviruses, potentially reducing pre-existing immunity issues in cattle

  • Potential targets include bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), and other BRDC pathogens

Co-formulation strategies:

• Combination vaccines:

  • Co-formulate recombinant HN proteins with antigens from other respiratory pathogens

  • Develop compatible adjuvant systems that enhance responses to all components

  • Balance antigen ratios to prevent immunodominance issues

• Universal epitope approaches:

  • Identify conserved epitopes across multiple paramyxoviruses

  • Engineer consensus antigens that induce cross-reactive immunity

  • Focus on conserved regions of viral attachment proteins

Applications in specific disease contexts:

• Bovine Respiratory Disease Complex (BRDC):

  • BPIV3 is one of several significant respiratory pathogens in BRDC

  • Multivalent vaccines targeting BPIV3, BRSV, and bacterial pathogens would address a critical need

  • rHAd5-F+HN could serve as a foundation for such multivalent approaches

• Human respiratory infections:

  • rB/HPIV3 expressing RSV antigens demonstrates the feasibility of creating bivalent human vaccines

  • Could be extended to include other human respiratory pathogens like metapneumovirus

  • The attenuation provided by BPIV3 backbone offers a built-in safety feature for human applications

• One Health applications:

  • Vaccines targeting pathogens with zoonotic potential

  • Combined protection against viruses affecting both livestock and humans

  • Reduction of viral reservoirs in animal populations to prevent spillover

The research foundation already exists for these applications, as demonstrated by the successful development of rB/HPIV3 expressing RSV glycoproteins and the effective immune responses generated by rHAd5-F+HN in both mice and calves . The approach of using BPIV3-derived vectors offers particular advantages in terms of safety, due to the host range restriction of BPIV3 in primates, while maintaining robust immunogenicity .