Laboratory And Pre-clinical Studies Of Parainfluenza Viruses

National Institute Of Allergy And Infectious Diseases

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Abstract

In recent years, we have focused on using a PIV3-based vector to express RSV antigen, providing a bivalent vaccine against the two most important pediatric viral respiratory pathogens. The vector is one that we previously developed, called rB/HPIV3, which consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3. This results in a chimeric virus that is attenuated in non-human primates and humans due to the BPIV3 backbone, and which bears the neutralization and major protective F and HN antigens of HPIV3. Both the empty B/HPIV3 vector and B/HPIV3 expressing the unmodified RSV F protein were previously shown to be well-tolerated in infants and young children. Therefore, this vector appears to be in the desired range of attenuation. In work continuing from previous years, we continued to focus on expressing the RSV fusion F glycoprotein because it generally is considered to be the most important RSV neutralization and protective antigen. RSV F also is much more highly conserved among RSV strains than the attachment G protein, which is the other neutralization antigen and the second most important protective antigen. We continued to evaluate a number of strategies to optimize the immunogenicity of rB/HPIV3 expressing RSV F protein. One modification was to increase the stability of the pre-fusion conformation of the F protein - the conformation that is the most effective in inducing RSV-neutralizing antibodies - by introducing mutations that have been reported by colleagues in the NIH Vaccine Research Center and elsewhere. The most successful mutations involved addition of a disulfide bond (called the DS mutation) in combination with two cavity-filling missense mutations (called Cav1). The other modification was to engineer RSV F to be efficiently packaged in the B/HPIV3 vector particle. This was done by replacing the transmembrane and cytoplasmic tail (TMCT) domains of RSV F with those of BPIV3 F. Each of these two modifications, DS-Cav1 and TMCT, resulted in a substantial increase in the induction of serum RSV-neutralizing antibodies, and in particular antibodies that neutralized RSV efficiently in vitro without added complement and thus are highly effective in neutralization. Clinical study material of these lead candidates has been prepared and characterized and will be evaluated in a Phase 1 study in 2025/2026. We continue to evaluate HPIV3 vaccine candidates that are attenuated by codon-pair deoptimization (CPD), in collaboration with Dr. Steffen Mueller and team (Codagenix, Inc). In open reading frames, certain synonymous codon pairs may be present in a species in a greater or lower abundance than statistically predicted, a phenomenon known as codon-pair bias. CPD relies on recoding of viral ORFs by substituting overrepresented codon pairs with synonymous but underrepresented pairs, which typically results in virus attenuation, but does not alter the protein composition of a virus. Thus, vaccine candidates attenuated by CPD express the full complement of viral proteins and antigenic sites. To attenuate HPIV3, the HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination by CPD. This resulted in 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. We found that CPD of N or L severely reduced growth in vitro, and these viruses were not further evaluated at that time. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. HPIV3 with CPD of HN and F yielded promising vaccine candidates suitable for further development. Further studies of HPIV3 attenuated by CPD are ongoing, with publication of study results expected in FY 2026.