Live-attenuated pediatric parainfluenza vaccine expressing 6P-stabilized SARS-CoV-2 spike protein is protective against SARS-CoV-2 variants in hamsters

The pediatric live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3)-vectored vaccine expressing the prefusion-stabilized SARS-CoV-2 spike (S) protein (B/HPIV3/S-2P) was previously evaluated in vitro and in hamsters. To improve its immunogenicity, we generated B/HPIV3/S-6P, expressing S further stabilized with 6 proline mutations (S-6P). Intranasal immunization of hamsters with B/HPIV3/S-6P reproducibly elicited significantly higher serum anti-S IgA/IgG titers than B/HPIV3/S-2P; hamster sera efficiently neutralized variants of concern (VoCs), including Omicron variants. B/HPIV3/S-2P and B/HPIV3/S-6P immunization protected hamsters against weight loss and lung inflammation following SARS-CoV-2 challenge with the vaccine-matched strain WA1/2020 or VoCs B.1.1.7/Alpha or B.1.351/Beta and induced near-sterilizing immunity. Three weeks post-challenge, B/HPIV3/S-2P- and B/HPIV3/S-6P-immunized hamsters exhibited a robust anamnestic serum antibody response with increased neutralizing potency to VoCs, including Omicron sublineages. B/HPIV3/S-6P primed for stronger anamnestic antibody responses after challenge with WA1/2020 than B/HPIV3/S-2P. B/HPIV3/S-6P will be evaluated as an intranasal vaccine to protect infants against both HPIV3 and SARS-CoV-2.

Introduction SARS-CoV-2 infects and causes disease in all age groups, including infants and children [1]. Even though clinical disease is generally mild in the pediatric population, the overall burden of COVID-19 in this population is substantial [2,3]. Effective SARS-CoV-2 vaccines are needed for all age groups, including children under 5 years of age. mRNA-based COVID-19 vaccines are available under emergency use authorization for children 6 months to 5 years of age. These and other injectable vaccines are designed to induce strong systemic immune responses [4][5][6]. However, SARS-CoV-2 is a respiratory virus, which infects and spreads via the respiratory mucosal route. Vaccines with the ability to induce mucosal immunity at the respiratory mucosal entry site are needed [7]. To this end, we are using a live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3) as a viral vector for intranasal immunization. Recombinant B/ HPIV3 was originally developed as a live-attenuated vaccine candidate for intranasal immunization against human parainfluenza virus type 3 [HPIV3] [8,9]. HPIV3 is an important pediatric respiratory pathogen in humans, second only to human respiratory syncytial virus (RSV) as a cause of severe lower respiratory tract disease in children under 5 years of age. HPIV3 circulates globally, causing annual epidemics, and represents an important vaccine target for the pediatric population [10][11][12]. The live-attenuated virus B/HPIV3 contains the N, P, M, and L genes of bovine parainfluenza virus (BPIV3), which is attenuated in primates [13]; the genes expressing the parainfluenza virus surface glycoproteins HN and F, the major neutralizing and protective antigens, were derived from HPIV3 [9]. This chimeric live-attenuated vaccine candidate against HPIV3 was immunogenic and well-tolerated in young children [8]. In previous studies, B/HPIV3 was also used as a vector to express the fusion (F) glycoprotein of another human respiratory pathogen, RSV, providing a bivalent HPIV3/RSV vaccine candidate [8,14]. A version of this vaccine candidate was well-tolerated in children >2 months of age [ [15], Clinicaltrials.gov NCT00686075], and improved versions are in further clinical development as bivalent pediatric vaccines against HPIV3 and RSV [16].
In a recent study, we introduced the wild-type and a prefusion-stabilized versions of SARS-CoV-2 spike (S) into the B/HPIV3 vector [17]. The immunogenicity and protective efficacy of these vaccine candidates against a SARS-CoV-2 challenge were evaluated in hamsters, a widely used animal model that supports robust replication of B/HPIV3 vectors and SARS-CoV-2 [18][19][20]. We found that a single intranasal dose of the B/HPIV3-vectored vaccine expressing the prefusion-stabilized version of SARS-CoV-2 S protein (B/HPIV3/S-2P) induced strong serum antibody responses to SARS-CoV-2 S and elicited serum antibodies with broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7/Alpha and B.1.351/Beta. While a secreted form of the S-2P stabilized version of the SARS-CoV-2 S protein is widely used in injectable mRNA vaccines and vectored or protein vaccines against COVID-19, a further-stabilized secreted version of S was developed, containing 4 additional proline substitutions [S-6P, [21]]. In transient transfection experiments, the expression level of S-6P was about 10-fold higher than that of S-2P, and the overall protein stability and resistance of the S-6P version towards heat stress, storage at room temperature, and multiple freeze-thaw cycles were increased, identifying the secreted version of S-6P as an improved version of S-2P [21].
We recently showed that B/HPIV3/S-6P is immunogenic and protective in rhesus macaques [22]. Here, we extend our work to (i) evaluate the effects of further stabilization by these four additional proline substitutions within the full-length version of the S protein on expression by B/HPIV3, and (ii) to compare the immunogenicity and protective efficacy of B/ HPIV3/S-2P and B/HPIV3/S-6P against vaccine-matched SARS-CoV-2 or Alpha and Beta variants in hamsters, and (iii) to compare the breadth of the SARS-CoV-2 antibody response induced by these two S-expressing B/HPIV3 vectors against variants of concern, including five Omicron sublineages.

Generation of B/HPIV3/S-6P and comparison of B/HPIV3/S-2P and B/HPIV3/S-6P in vitro
We previously used B/HPIV3 to express a prefusion-stabilized version of the full-length SARS-CoV-2 S protein (Wuhan-Hu-1, GenBank MN908947), codon-optimized for human expression, and with the S1/S2 polybasic furin cleavage motif "RRAR" ablated by aa substitutions (RRAR-to-GSAS; [23]), resulting in B/HPIV3/S-2P [17]. Here, we generated an additional version, B/HPIV3/S-6P, expressing a full-length version of the SARS-CoV-2 S protein that had been further stabilized by four additional proline substitutions (aa 817, 892, 899 and 942; [21], Fig 1A). For expression in B/HPIV3, the open reading frames (ORFs) encoding the prefusionstabilized S proteins were framed by nucleotide adapters containing the BPIV3 gene start and gene end signals for insertion as an additional gene between the BPIV3 N and P ORFs (Fig 1A).
B/HPIV3 expressing the S-6P prefusion-stabilized version of the S protein was readily recovered from cDNA by reverse genetics. The sequences of all recombinant B/HPIV3 viruses used in this study were confirmed by Sanger sequencing. We next compared the stability of S expression of S-2P and S-6P by dual-staining immunoplaque assay, performed with polyclonal hyperimmune antisera against PIV3 antigens (red pseudo-coloring), and against the recombinantly-expressed secreted version of the SARS-CoV-2 S-2P protein (green pseudo-coloring). B/HPIV3/S-2P plaques appeared heterogeneous in size, while B/HPIV3/S-6P plaques were more homogenous (Fig 1B). In virus stocks grown from eight independent recoveries of B/ HPIV3/S-2P or B/HPIV3/S-6P, staining for both PIV3 and SARS-CoV-2 S (yellow, Fig 1B) was obtained for 94.9±3.4% and 96.7±1.7% of plaques, respectively, indicating that both viruses stably expressed SARS-CoV-2 S protein. In Vero cells, which are widely used for vaccine manufacture, multicycle replication of B/HPIV3/S-2P and B/HPIV3/S-6P was similarly efficient and only slightly delayed compared to B/HPIV3 (Fig 1C), while in human lung epithelial A549 cells, replication of both B/HPIV3/S-2P and B/HPIV3/S-6P was reduced by about 10-fold compared to B/HPIV3 ( Fig 1D).
Next, we compared the expression of the SARS-CoV-2 S by B/HPIV3/S-2P and B/HPIV3/ S-6P by Western blotting. We infected Vero and A549 cells at a multiplicity of infection BPIV3 genes are shown in blue, HPIV3 genes encoding the fusion and hemagglutinin-neuraminidase proteins are in red, and the S gene is in orange. Each gene, including the SARS-CoV-2 S gene, begins and ends with PIV3 gene start (GS) and gene end (GE) transcription signals (light and dark grey bars, respectively). The S gene encodes a prefusion-stabilized uncleaved (S-2P) version of the S protein, or a further stabilized version (S-6P) with 6 proline substitutions [6P; [21]], and was inserted into an AscI site between the BPIV3 N and P genes [17]. The stabilizing proline substitutions and four aa substitutions that ablate the furin cleavage site (RRAR to GSAS, aa 682-685) in the S-2P and S-6P proteins are indicated. (B) Stability of SARS-CoV-2 expression, analyzed by dual-staining immunoplaque assay. Virus stocks were titrated on Vero cells and analyzed by dual-staining immunoplaque assay essentially as described [17], using a goat hyperimmune antiserum against a recombinantly-expressed secreted form of S-2P protein and a rabbit hyperimmune antiserum against HPIV3 virions. HPIV3-and SARS-CoV-2 S-specific staining was pseudocolored in red and green, respectively; dual staining appeared as yellow. The percentage (± standard deviation) of yellow plaques is indicated at the bottom. (C, D) Multicycle replication of B/HPIV3 vectors on Vero and human lung epithelial A549 cells. Cells in 6-well plates were infected in triplicate with indicated viruses at a multiplicity of infection (MOI) of 0.01 PFU per cell and incubated at 32˚C for a total of 7 days. At 24 h intervals, aliquots of culture medium were collected and flash-frozen for subsequent immunoplaque titration on Vero cells. (E, F, G) Viral proteins in infected cell lysates (E, F) and purified virions (G). Vero (E) or A549 (F) cells in 6-well plates were infected with B/HPIV3, B/HPIV3/S-2P or B/HPIV3/S-6P at an MOI of 1 and incubated at 32˚C for 48 h. Cell lysates were prepared and analyzed by SDS-PAGE under denaturing and reducing conditions and Western blotting. The SARS-CoV-2 S protein was detected using the goat hyperimmune serum to S-2P protein, and the BPIV3 N protein was detected by a rabbit hyperimmune serum raised (MOI) of 0.1 plaque-forming unit (PFU) per cell with B/HPIV3 or the S-expressing versions, and prepared cell lysates 24 h after infection. Following SDS-PAGE (under reducing and denaturing conditions) and Western blotting using the hyperimmune antiserum against the secreted version of SARS-CoV-2 S-2P or purified HPIV3 virions, we detected the S protein in lysates at a size consistent with the uncleaved S0 precursor protein (Fig 1E and 1F).
We also evaluated the incorporation of the SARS-CoV-2 S-2P or S-6P protein into B/HPIV3 particles. Vero cell-grown viruses were purified by ultracentrifugation through sucrose gradients in three independent experiments. Western blotting revealed that both the S-2P and S-6P versions of the S protein were incorporated in B/HPIV3 particles (Fig 1G).

Following intranasal administration, B/HPIV3/S-2P and B/HPIV3/S-6P replicate efficiently in hamsters
To evaluate the replication of B/HPIV3/S-2P and B/HPIV3/S-6P in the hamster model, groups of 28 hamsters were immunized intranasally with 5.0 log 10 PFU of B/HPIV3/S-2P, B/HPIV3/ S-6P, or B/HPIV3 empty vector (Experiment #1, Fig 2A). On days 3, 5, and 7 post-immunization (pi), 5 hamsters per group were euthanized to evaluate vaccine virus replication in the respiratory tract. Nasal turbinates (NT) and lung tissue homogenates were prepared, and vaccine virus titers were determined by immunoplaque assay (Fig 2B-2C). Additional hamsters from each group were euthanized on days 3 (n = 1) and 5 (n = 2), and lungs and NT were analyzed by immunohistochemistry (IHC) (Fig 2D and 2E). Sera were obtained on day 26 pi from 10 animals per group to evaluate the antibody response.
Consistent with our previous results [17], B/HPIV3 replicated well in NT and lungs, with highest titers on day 3 pi (6.5 and 5.9 log 10 PFU/g, respectively) ( Fig 2B, 2C). By day 5 pi, B/ HPIV3 titers were 20-fold lower in NT, and about 10-fold lower in lungs. B/HPIV3/S-2P and B/HPIV3/S-6P also replicated well. On day 3 pi, titers of B/HPIV3/S-2P and B/HPIV3/S-6P in NT were 30 and 125-fold lower, respectively, compared to the B/HPIV3 empty vector; titers in lungs were 100-and 160-fold lower, respectively, than those of the B/HPIV3 empty vector. By day 5 pi, titers of B/HPIV3/S-2P and B/HPIV3/S-6P had increased in NT (25-and 50-fold, respectively) and lungs (6-and 20-fold, respectively), consistent with delayed replication in the upper and lower respiratory tract due to the presence of the insert. By day 7 pi, replication of B/HPIV3/S-2P, B/HPIV3/S-6P, and empty vector was very low or, in some animals, undetectable in NT or lungs, reflecting self-limiting replication of the vaccine candidates. We also determined the stability of S-2P and S-6P protein expression during in vivo replication by dual-staining immunoplaque assay; 96% of B/HPIV3/S-2P and 98% of B/HPIV3/S-6P plaques from NT and 95% of B/HPIV3/S-2P and 99% of B/HPIV3/S-6P plaques from lungs obtained on day 3 after immunization were positive for S protein; 90% and 98% (NT) or 87% and 96% (lungs) of B/HPIV3/S-2P and B/HPIV3/S-6P plaques from specimens obtained on day 5 after immunization were positive for the S protein, showing that expression of both versions of the S protein was stably maintained in vivo.
We also evaluated antigen expression in the NT and lungs by IHC. Representative IHC images from NT and lungs obtained on day 5 pi from one of two animals per group are shown in Fig 2D and 2E. B/HPIV3 antigen was present in columnar epithelial cells lining the nasal against sucrose-purified HPIV3, followed by immunostaining with infrared fluorophore labeled secondary antibodies and infrared imaging. Immunostaining for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a loading control. Images were acquired and analyzed using Image Studio software (LiCor) and are representatives of three independent experiments. (G) Vero-grown virus preparations of B/HPIV3, B/HPIV3/S-2P, and B/HPIV3/S-6P were purified by centrifugation through 30/60% sucrose gradients, and gently pelleted by centrifugation to remove sucrose. One μg of protein per lane was used for SDS-PAGE and Western blotting as described above. Ladder, molecular size marker. https://doi.org/10.1371/journal.ppat.1011057.g001

PLOS PATHOGENS
B/HPIV3/S-6P is protective against SARS-CoV-2 variants in hamsters In Experiment #1, six-week-old golden Syrian hamsters in groups of 28 were inoculated intranasally with 5 log 10 PFU of the indicated viruses. On days 3, 5, and 7, five animals per group were sacrificed and the viral titers in the nasal turbinates (NT) (B) and lungs (C) were determined by dualstaining immunoplaque assay. Titers from individual animals are represented by symbols, and geometric mean titers (GMT) and standard deviations are shown by lines. GMT values are indicated below the dotted line; the maximum mean peak titer irrespective of day for each group is in bold. The limit of detection (LOD) was 50 PFU/g of tissue (dotted line). * = P<0.05; ** = P<0.01; *** = P<0.001; **** = P<0.0001 (Two-way ANOVA with Tukey's multiple comparisons). (D, E) NT (D) and lung tissues (E) obtained on day 5 (n = 2 animals per group and n = 1 uninfected control animal) were processed for immunohistochemistry. Serial sections were immunostained for HPIV3 and SARS-CoV-2 antigen using hyperimmune antisera raised against HPIV3 virions and a secreted form of the S-2P protein, respectively. Lung sections (E) include bronchi (larger airways, characterized by pseudostratified layers of epithelial cells) or bronchioli (smaller airways, lined with a single layer of PLOS PATHOGENS B/HPIV3/S-6P is protective against SARS-CoV-2 variants in hamsters mucosa and the bronchioles in tissues from B/HPIV3, B/HPIV3/S-2P, and B/HPIV3/S-6Pimmunized animals (red arrowheads, Fig 2D and 2E). SARS-CoV-2 S antigen in animals immunized with B/HPIV3/S-2P and B/HPIV3/S-6P similarly was present in columnar airway epithelial cells of NT and bronchioles (blue arrowheads, Fig 2D and 2E). The B/HPIV3 and SARS-CoV-2 S immunostaining patterns and intensities in tissues of B/HPIV3/S-2P and B/ HPIV3/S-6P-immunized animals were generally similar. Thus, B/HPIV3/S-2P and B/HPIV3/ S-6P efficiently infected and expressed the SARS-CoV-2 S protein in columnar epithelial cells of the nasal and bronchial mucosa.

B/HPIV3/S-2P and B/HPIV3/S-6P elicited strong serum anti-S and anti-RBD IgG and IgA responses in hamsters
To evaluate immunogenicity and protection against SARS-CoV-2 challenge, 45 hamsters per group were immunized intranasally with 5.0 log 10 PFU of B/HPIV3/S-2P, B/HPIV3/S-6P, or B/HPIV3 empty vector (Experiment #2, Fig 3A). We evaluated serum antibody responses 26 or 27 days after intranasal immunization. First, we measured SARS-CoV-2 S-specific serum IgG ( Fig 3B) and IgA ( Fig 3C) by ELISA using as antigens the secreted form of the S-2P protein (left panels) and a fragment (aa 319-591) of the S protein bearing the receptor-binding domain (RBD) (right panels). Intranasal immunization with B/HPIV3/S-2P and B/HPIV3/S-6P elicited strong serum anti-S and anti-RBD IgG and IgA responses in hamsters; the geometric mean (GMT) anti-S and anti-RBD IgG titers in the B/HPIV3/S-6P group were significantly higher (1.5-fold, P<0.01, and 2.4-fold, P<0.001) than those induced by B/HPIV3/S-2P. The GMT anti-RBD IgA titers in the B/HPIV3/S-6P group were also significantly higher than those induced by B/HPIV3/S-2P (1.4-fold, P<0.05), while the difference in anti-S IgA titers did not reach the level of significance. We also evaluated the serum antibody response in animals from Experiment #1 (n = 10 per group, S1A Fig), confirming that B/HPIV3/S-6P elicited higher anti-RBD IgG and IgA responses (S1B and S1C Fig; P<0.05). In Experiment #1, the differences in the anti-S IgG and IgA responses between B/HPIV3/S-2P and B/HPIV3/S-6P did not reach the level of statistical significance (S1B and S1C Fig).
We also determined SARS-CoV-2 challenge virus titers after challenge with strain WA1/ 2020 or VoCs (B.1.1.7/Alpha or B.1.351/Beta) in tissue homogenates of NT and lung tissues obtained on days 3 and 5 from 5 animals per group (Fig 5A-5C). After challenge of B/HPIV3 empty-vector immunized animals with WA1/2020, B.1.1.7/Alpha or B.1.351/Beta variants, we detected high SARS-CoV-2 titers in lungs on both days, with peak GMTs of 8.0 log 10 TCID 50  Next, we determined the SARS-CoV-2 viral loads in lungs after challenge. RNA from lung homogenates was evaluated by TaqMan assays to detect genomic N (gN) and E (gE) SARS-CoV-2 RNA, as well as subgenomic E (sgE) RNA. sgE RNA served as an indicator for active replication of challenge virus (Fig 5D-5F). In tissues obtained from all B/HPIV3 empty-vector immunized animals on days 3 or 5 after SARS-CoV-2 challenge, we detected very high viral loads (ranging between 9 and 11 log 10 copies per g) of gN, gE, and sgE per g of lung tissue. The high levels of sgE copy numbers confirmed high levels of WA1/2020, B.
We also evaluated the ability of the sera from the challenged animals to block binding of a soluble version of ACE2 to S proteins derived from various SARS-CoV-2 lineages, using the ACE2 binding inhibition assay as a high-throughput alternative to BSL3 virus neutralization assays. Twenty-one days after challenge with WA1/2020, B.1.1.7 or B.1.351, sera from B/ HPIV3 empty-vector control immunized/SARS-CoV-2 challenged animals inhibited ACE2

PLOS PATHOGENS
B/HPIV3/S-6P is protective against SARS-CoV-2 variants in hamsters binding of S proteins from all 15 lineages and sublineages tested, showing that in the emptyvector control animals, each of the three challenge viruses induced primary antibody responses with broad ACE2 binding inhibition activity to S proteins. Specifically, at the dilution tested, post-challenge sera from B/HPIV3 empty-vector immunized animals completely blocked ACE2 binding to S proteins of the challenge virus-matched lineages (Fig 6B, top panels, red labels), and to heterologous WA1/2020 and B.1.1.7/Alpha-derived S proteins (Figs 6B and S3, black labels); ACE2 binding inhibition by these sera to the B.1.617.2/Delta-derived S protein also was nearly complete, while the median ACE2 binding inhibition of post-challenge sera from B/HPIV3 control-immunized animals to S proteins of Omicron variants ranged between 45 and 90% (Figs 6B and S3).

Discussion
Injectable SARS-CoV-2 vaccines provide substantial protection against COVID-19-induced hospitalization and death. However, protection against SARS-CoV-2 infection and shedding, as well as against symptomatic infection, is short-lived, particularly against Omicron variants [25][26][27][28]. Ideally, a SARS-CoV-2 vaccine should induce a strong immune response in the upper and lower airways to restrict SARS-CoV-2 replication at the entry site of infection, with the ability to prevent disease. Intranasal vector vaccines will be useful to complement injectable SARS-CoV-2 vaccines for all age groups due to their ability to induce robust protection against SARS-CoV-2 infection and replication in the respiratory tract. The live-attenuated B/HPIV3 vector described in this study has been well-characterized in previous pediatric clinical studies as a pediatric vaccine candidate for intranasal immunization and was safe and immunogenic in infants as young as 2 months of age [8,15]. In a previous study, we showed that a B/HPIV3 vector expressing the SARS-CoV-2 S protein from the original Wuhan-Hu-1 isolate that was stabilized in the prefusion form (B/HPIV3/S-2P) did not induce any weight loss, and was immunogenic and protective in hamsters [17]. In the present study, we aimed to (i) further improve the immunogenicity of B/HPIV3/S-2P, (ii) evaluate the protection conferred by the S-expressing B/HPIV3 vectors against challenge with SARS-CoV-2 from two heterologous lineages in hamsters, and (iii) evaluate the magnitude and breadth of the antibody response of immunized hamsters following challenge with the homologous or heterologous SARS-CoV-2 isolates.
In our previous study [17], we used the "S-2P" version as a SARS-CoV-2 vaccine antigen. This S ORF corresponds to the first available SARS-CoV-2 sequence in which the furin cleavage site "RRAR" was replaced by the "GSAS" motif and two proline substitutions at aa 986 and 987 were introduced to stabilize S into its prefusion form. The resulting B/HPIV3/S-2P was highly immunogenic and protective against a challenge with the homologous SARS-CoV-2 WA1/2020 isolate [17]. However, a study showed that four additional proline substitutions at PLOS PATHOGENS B/HPIV3/S-6P is protective against SARS-CoV-2 variants in hamsters aa positions 817, 892, 899 and 942 of S introduced into a soluble prefusion-stabilized version of the S protein (S-6P) significantly increased the level of transient expression of S from plasmids in transfected human cells (six to 10-fold), and increased the physical stability of the S protein without affecting its antigenicity [21]. We hypothesized that the potentially increased expression of S-6P compared to S-2P by the B/HPIV3 vector might increase the S-specific immune response. Even though we were not able to detect a statistically significant increase in expression of S-6P over S-2P by B/HPIV3 in Vero or A549 cells, S-6P seemed to be incorporated into Vero-grown B/HPIV3 particles more efficiently than S-2P. Previously, we found that the incorporation of the RSV fusion (F) protein into B/HPIV3 virions increased the magnitude and quality of serum RSV-neutralizing antibodies [16,29]. We speculated that the increased immunogenicity associated with incorporation into the vector particle could be due to more efficient presentation of the heterologous antigen. In the present study, we found in two independent hamster experiments that B/HPIV3/S-6P induced significantly higher serum anti-RBD IgG and IgA antibody titers than B/HPIV3/S-2P, suggesting that B/HPIV3/S-6P was modestly but significantly more immunogenic than B/HPIV3/S-2P. This might be due to the improvements in the structure-based design, resulting in increased physical stability of the S-6P version [21], and/or the modest increase in incorporation in the B/HPIV3 particles observed in this study.
We also evaluated the breadth of neutralization conferred by the S-expressing B/HPIV3 vectors against VoCs. Serum antibodies from B/HPIV3/S-2P and B/HPIV3/S-6P-immunized hamsters efficiently neutralized the homologous SARS-CoV-2 WA1/2020 isolate as well as VoCs of lineage B.1.1.7/Alpha, B.1.351/Beta or B.1.617.2/Delta. In an ACE2 binding inhibition assay, used as a surrogate for BSL3 virus neutralization assays, hamster sera were less efficient in inhibiting ACE2 binding to S derived from B.1.1.529/Omicron sublineages than from WA1/2020 or other VoCs. However, sera from B/HPIV3/S-2P-and B/HPIV3/S-6P-immunized hamsters were still able to substantially inhibit ACE2 binding of S derived from Omicron sublineages. Thus, a single intranasal immunization with the S-expressing B/HPIV3 vectors induced a broad neutralization to antigenically distinct SARS-CoV-2 variants.
B/HPIV3/S-2P and B/HPIV3/S-6P-immunized hamsters were protected from weight loss and lung inflammation following homologous or heterologous (B.1.1.7/Alpha and B.1.351/ Beta lineages) SARS-CoV-2 challenge. Only low levels of B.1.351/Beta replication were detected in the NT and lung of B/HPIV3/S-2P and B/HPIV3/S-6P-immunized hamsters that was cleared by day 5 post-challenge. Our results suggested that a single intranasal immunization with B/HPIV3/S-2P or B/HPIV3/S-6P conferred a broad protection in both the upper and lower airways against at least the Alpha and Beta VoCs. Thus, protection induced in the respiratory tract by these vectors seemed to be superior to injectable vaccines such as the adenovirus-based vaccines or alphavirus-based replicating RNA vaccines which in the hamster model were less effective in reducing SARS-CoV-2 challenge virus loads in the lungs [30] or the upper airways of immunized hamsters following challenge with VoCs [31,32].
Three weeks after challenge, B/HPIV3/S-2P and B/HPIV3/S-6P-immunized hamsters exhibited anamnestic serum antibody responses with an increased breadth of neutralizing activities against all tested SARS-CoV-2 lineages, including 10 different B.1.1.529/Omicron sublineages. Furthermore, B/HPIV3/S-6P appeared to prime for a stronger anamnestic antibody response than B/HPIV3/S-2P, inducing three-fold higher anamnestic neutralizing antibody titers against the homologous SARS-CoV-2 isolate. Since B/HPIV3/S-6P induced more robust primary serum IgA and IgG responses and primed for stronger and broader anamnestic responses, B/HPIV3/S-6P was further evaluated in rhesus macaques [22]. Based on its broad immunogenicity, and the efficacy against two SARS-CoV-2 variants described here, and based on the strong systemic and mucosal immunogenicity in macaques described previously [22], B/HPIV3/S-6P is currently being advanced to a phase I study as an intranasal live attenuated vaccine against both HPIV3 and SARS-CoV-2 for infants and young children.
In this study, we evaluated B/HPIV3 as an intranasal vector vaccine candidate expressing the SARS-CoV-2 S protein from an additional gene. B/HPIV3 is attenuated in humans by the N, P, M, and L genes, which are derived from bovine parainfluenza virus type 3. Thus, B/ HPIV3 is expected to be safe in HPIV3 seronegative infants and children and suitable as a bivalent vaccine against HPIV3 and SARS-CoV-2, but it will likely be over-attenuated in HPI-V3-experienced individuals. In the best-case scenario, intranasal immunization of HPIV3-experienced individuals with B/HPIV3 expressing the prefusion-stabilized SARS-CoV-2 S antigen would confer a protective mucosal immune response to SARS-CoV-2 due to the high level of immunogenicity of the prefusion-stabilized S protein, and despite the greatly restricted ability of HPIV3 to replicate in the respiratory tract in presence of pre-existing anti-vector immunity. We are planning to evaluate in detail the mucosal immune responses in primeboost studies in animal models. In another limitation, in this study, we characterized the differences in prefusion stabilization of two S antigens derived from the ancestral Wuhan-Hu-1 isolate, but the currently circulating SARS-CoV-2 variants have evolved to accumulate antigenic differences from the ancestral strain. However, our results show that the breadth of protection and antibody reactivity to antigenically distant variants are substantial. We are currently including S antigens from other variants in our studies to characterize the differences in immune responses and protective efficacy of vaccine-matched vs non-matched antigens in the hamster challenge model.

Ethics statement
All animal studies were approved by the NIAID Animal Care and Use Committee.

Dual-staining immunoplaque assay
Briefly, Vero cells in 24-well plates were inoculated with 10-fold serially diluted samples. Two hours later, monolayers were overlaid with culture medium containing 0.8% methylcellulose and incubated at 32˚C. Six days later, monolayers were fixed with 80% methanol, and immunostained with an HPIV3-specific rabbit hyperimmune serum to detect B/HPIV3 antigens, and a goat hyperimmune serum against the secreted version of SARS-CoV-2 S-2P to detect co-expression of the S protein. An infrared-dye conjugated donkey anti-rabbit IRDye680 IgG and a donkey anti-goat IRDye800 IgG were used as secondary antibodies. Stained plates were scanned with the Odyssey infrared imaging system (LiCor). Fluorescent staining for PIV3 proteins and SARS-CoV-2 S was visualized in green and red, respectively, generating yellow plaque staining when merged [17].

Multicycle replication of B/HPIV3 vectors in cell culture
Sub-confluent Vero and A459 cells in 6-well plates were infected in triplicate with indicated viruses at a multiplicity of infection (MOI) of 0.01 PFU per cell. After adsorption for 2 h, cells were washed to remove the virus inoculum, and 3 ml of fresh media were added. Cells were incubated at 32˚C for 7 days. Every 24 h, 0.5 ml aliquots of culture medium were collected and snap-frozen in dry ice, and 0.5 ml of fresh medium was added to each well. Virus aliquots from each time point were titrated side-by-side on 24-well plates of Vero cells as described above.

SDS-PAGE and Western blot analysis
Six-well plates of Vero or A549 cells were inoculated using an MOI of 1 PFU per cell with B/ HPIV3, B/HPIV3/S-2P, and B/HPIV3/S-6P and incubated at 32˚C. At 48 h post-inoculation, cells were washed once with cold PBS and lysed using 300 μl LDS lysis buffer (Thermo Fisher Scientific) containing NuPAGE reducing reagent (Thermo Fisher Scientific). Cell lysates were further passed through QIAshredder columns (Qiagen), heated for 10 min at 95˚C and separated on 4-12% Bis-Tris NuPAGE gels (Thermo Fisher Scientific) in the presence of antioxidant (Thermo Fisher Scientific). The resolved proteins were transferred to polyvinylidene difluoride membranes which were blocked with blocking buffer (LiCor) for 1 h at room temperature. Then, blocked membranes were incubated with a goat hyperimmune serum to SARS-CoV-2 S and a rabbit polyclonal hyperimmune sera against purified HPIV3 in blocking buffer overnight at 4˚C. A mouse monoclonal antibody to GAPDH (Sigma) was included as a loading control. Membranes were incubated with infrared dye-labeled secondary antibodies (donkey anti-rabbit IgG IRDye 680, donkey anti-goat IgG IRDye 800 or IRDye 680, and donkey anti-mouse IgG IRDye 800, LiCor). Intensities of individual protein bands from the acquired images were quantified after background correction using Image Studio software (LiCor).
The protein composition of virus particles was also analyzed. To do so, viruses were grown on Vero cells and the supernatant was purified by centrifugation through 30%/60% sucrose gradients. The sucrose-purified virus preparations were gently pelleted by centrifugation to remove sucrose as described previously [34]. The protein concentration of the purified virus preparations was determined using a BCA protein assay kit (Thermo Fisher Scientific) prior to the addition of lysis buffer. One μg of protein per lane was used for SDS-PAGE and Western blotting.

Replication, immunogenicity, and protective efficacy against SARS-CoV-2 challenge in hamsters
In Experiment #1, groups (n = 28) of 5-to 6-week-old male golden Syrian hamsters (Envigo Laboratories, Frederick, MD) were anesthetized and inoculated intranasally with 100 μl of Leibovitz's L-15 medium (Thermo Fisher Scientific) containing 5 log 10 PFU of B/HPIV3, B/ HPIV3/S-2P, or B/HPIV3/S-6P viruses. On days 3, 5, and 7 post-inoculation, 5 hamsters per group were euthanized by CO 2 inhalation, and nasal turbinates and lungs were collected to evaluate virus replication. NT and lung tissue samples for histology were obtained from one and two additional hamsters per group on days 3 and 5, respectively. For quantification of virus replication, tissues were homogenized in Leibovitz's L-15 medium, and clarified homogenates were analyzed by dual-staining immunoplaque assay on Vero cells as described above. On day 26 post-immunization, sera were collected from the remaining 10 animals per group to evaluate the immunogenicity of the vaccine candidates. HPIV3 vector-specific neutralizing antibodies were detected by a 60% plaque reduction neutralization test (PRNT 60 ) [35] on Vero cells in 24-well plates using HPIV3 expressing the eGFP protein. To determine the serum neutralizing antibody response to SARS-CoV-2, two-fold dilutions of heat-inactivated hamster sera were tested in a microneutralization assay for the presence of antibodies that neutralized the replication of 100 TCID 50 of SARS-CoV-2 in Vero E6 cells as previously described [17]. Serum IgG antibodies to a secreted form of the S-2P protein [23] or its RBD [36] were measured by ELISA [17], and serum IgA antibodies to the secreted form of S-2P or RBD were measured by europium ion-enhanced DELFIA-TRF assay (Perkin Elmer) following the supplier's protocol.
In Experiment #2, groups (n = 45) of 5-to 6-week-old male golden Syrian hamsters were immunized intranasally with 5 log 10 PFU of B/HPIV3, B/HPIV3/S-2P, or B/HPIV3/S-6P as described above. Sera were obtained on days 26 or 27. Hamsters in each immunization group were randomly distributed to 3 challenge virus subgroups of 15 animals per subgroup, and challenged intranasally with 4.5 log 10 TCID 50 of SARS-CoV-2 WA1/2020, the USA/ CA_CDC_5574/2020 isolate (lineage B.1.1.7/Alpha variant) or USA/MD-HP01542/2021 (lineage B.1.351/Beta variant) in 100 μl. Five hamsters from each challenge virus subgroup were euthanized by CO 2 inhalation on days 3 and 5 after challenge, and tissues were collected to evaluate challenge virus replication. The presence of challenge virus in clarified tissue homogenates was evaluated later by TCID 50 assay on Vero E6 cells. The remaining 5 hamsters from each challenge virus subgroup were euthanized on day 21 after challenge, and sera were collected for antibody analysis.

Immunohistopathology analysis
Lung tissue samples from hamsters were fixed in 10% neutral buffered formalin, processed through a Leica ASP6025 tissue processor (Leica Biosystems), and embedded in paraffin. Five μm tissue sections were stained with hematoxylin and eosin for routine histopathology. For IHC evaluation, sections were deparaffinized and rehydrated. After epitope retrieval, sections were labeled with a goat hyperimmune serum to anti-SARS-CoV-2 S (N25-154) at 1:1000, and a rabbit polyclonal anti-HPIV3 serum [37] at 1:500. Chromogenic staining was carried out on the Bond RX platform (Leica Biosystems) according to manufacturer-supplied protocols. Detection with DAB chromogen was completed using the Bond Polymer Refine Detection kit (Leica Biosystems). The VisUCyte anti-goat horseradish peroxidase (HRP) polymer (R&D Systems) replaced the standard Leica anti-rabbit HRP polymer from the kit to bind the goat anti-SARS-CoV-2 S antibodies. Slides were finally cleared through gradient alcohol and xylene washes prior to mounting. Sections were examined by a board-certified veterinary PLOS PATHOGENS B/HPIV3/S-6P is protective against SARS-CoV-2 variants in hamsters pathologist using an Olympus BX51 light microscope. Images were acquired using an Olympus DP73 camera.

Lentivirus-based pseudotype virus neutralization assay
Single-round luciferase-expressing pseudoviruses were generated by co-transfecting plasmids encoding SARS-CoV-2 S of isolate B.1.617.2/Delta or B.1.1.529/Omicron, luciferase reporter (pHR' CMV Luc), lentivirus backbone (pCMV ΔR8.2), and human transmembrane protease serine 2 (TMPRSS2) at a ratio of 1:20:20:0.3 into HEK293T/17 cells (ATCC CRL-11268) using transfection reagent LiFect293. Three days post-transfection, supernatants were collected and centrifugated at 478 x g for 10 min to remove cell debris and filtered through a 0.45 μm filter. Pseudoviruses were then aliquoted and titrated. For the antibody neutralization assay, 6-point, 5-fold dilution series were prepared in DMEM medium supplemented with 10% FBS, 1% Pen/ Strep and 3 μg/ml puromycin. Fifty μl of the diluted sera were mixed with 50 μl of diluted pseudoviruses in 96-well plates and incubated for 30 min at 37˚C. Then, ten thousand of ACE2-expressing 293T-cells (293T-hACE2.MF stable cell line) per well were added. Three days later, cells were lysed with Bright-Glo luciferase assay substrate (Promega), and luciferase activity (relative light units, RLU) was evaluated. The percent neutralization was normalized relative to uninfected cells as 100% neutralization and cells infected with only pseudoviruses as 0% neutralization. Fifty percent inhibitory concentration (IC 50 ) titers were determined using a log (agonist) vs. normalized response (variable slope) nonlinear function in Prism v9 (GraphPad). In brief, plates were blocked with MSD blocker A for 30 min and washed with MSD wash buffer. Heat-inactivated hamster sera were diluted 1:20 in diluent and added to duplicate wells. After a one-hour incubation, Sulfo-tag labelled ACE2 was added to each well. All incubations were performed on a plate shaker at room temperature. Following a one-hour incubation, plates were washed, MSD GOLD electrochemiluminescence read buffer B was added, and chemiluminescence of bound Sulfo-tag-ACE2 was detected using a MESO QuickPlex SQ 120 reader (MSD). The average electrochemiluminescence signals in duplicate wells for each serum were determined, as well as the maximum electrochemiluminescence signals of ACE2/S protein binding in no-serum control wells. The ACE2 neutralizing activity of each serum is represented as percent inhibition relative to no-serum controls.

RT-qPCR analysis of gene expression in lung tissue
Total RNA was extracted from 0.1 ml of lung homogenates (0.1 g/ml) using the TRIzol LS Reagent and Phasemaker Tubes Complete System (Thermo Fisher) along with the PureLink RNA Mini Kit (Thermo Fisher) following the manufacturer's instructions, and eluted in 0.1 ml RNase-free water. Total RNA was also extracted from lung homogenates of five control hamsters (non-immunized and non-challenged) in the same manner. cDNA was synthesized PLOS PATHOGENS B/HPIV3/S-6P is protective against SARS-CoV-2 variants in hamsters from 7 μl of RNA using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher). TaqMan assays (Thermo Fisher) specific for hamster (Mesocricetus auratus) CXCL10, Mx2, or IFN-L [38,39] were performed using the TaqMan Fast Advanced Master Mix (Thermo Fisher), and hamster beta-actin was included as a housekeeping gene. qPCR results were analyzed using the comparative threshold cycle (ΔΔC T ) method, normalized to beta-actin, and expressed as fold changes over the average expression of five uninfected, unchallenged hamsters.
To detect viral genomic N (gN), E (gE) RNA, and subgenomic E (sgE) mRNA of SARS-CoV-2 challenge virus WA1/2020, B.1.1.7/Alpha, or B.1.351/Beta in the lung homogenates, cDNA was synthesized from 8 μl of total RNA extracted as described above and TaqMan qPCRs for genomic N, E, and sgE were performed using previously-described primers and probes [40][41][42] and the TaqMan RNA-to-Ct 1-Step Kit (Thermo Fisher). Assays were performed on a QuantStudio 7 Pro real-time PCR system (Thermo Fisher). Standard curves were generated using serially diluted pcDNA3.1 plasmids containing gN, gE, or sgE sequences. The sensitivity of the TaqMan assay was 200 copies, corresponding to a limit of detection of 5.4 log 10 copies per g of tissue.

Statistical analysis
Data sets were assessed for significance using one-way ANOVA with Tukey's or Sidak's multiple comparisons test, two-way ANOVA with Tukey's post-hoc test or Mixed-effects analysis with Tukey's post-hoc test using Prism 9 (GraphPad Software). Data were only considered significant at P � 0.05. Details on the statistical comparisons can be found in the figures, figure legends, and results. Values of n are provided in the text and in the figure legends.
The numerical data used in all figures are included in S1 Data.