A single intranasal dose of a live-attenuated parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in hamsters

Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2–neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 104.5 50% tissue-culture infectious-dose (TCID50) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 106.6 TCID50/g in lungs and 107 TCID50/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P–immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2.

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Protective Antibodies Against Human Parainfluenza Virus Type 3 (HPIV3) Infection

10.1101/2020.06.15.153478 ◽

2020 ◽

Author(s):

Jim Boonyaratanakornkit ◽

Suruchi Singh ◽

Connor Weidle ◽

Justas Rodarte ◽

Ramasamy Bakthavatsalam ◽

...

Keyword(s):

Virus Type ◽

Neutralizing Antibodies ◽

Parainfluenza Virus ◽

Respiratory Pathogen ◽

Effective Vaccine ◽

Healthy Children ◽

Protective Antibodies ◽

Human Parainfluenza Virus ◽

Type 3

ABSTRACTHuman parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. Unfortunately, an effective vaccine or therapeutic is not currently available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, or spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3 neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Importantly, prophylactic administration of one of these antibodies, named PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.

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Mechanism of Interference Mediated by Human Parainfluenza Virus Type 3 Infection

Journal of Virology ◽

10.1128/jvi.74.24.11792-11799.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11792-11799 ◽

Cited By ~ 21

Author(s):

Maria-Arantxa Horga ◽

G. Luca Gusella ◽

Olga Greengard ◽

Natalia Poltoratskaia ◽

Matteo Porotto ◽

...

Keyword(s):

Virus Type ◽

Fluorescent Protein ◽

Parainfluenza Virus ◽

Wild Type ◽

Challenge Virus ◽

Neuraminidase Activity ◽

Definitive Evidence ◽

Viral Interference ◽

Human Parainfluenza Virus ◽

Type 3

ABSTRACT Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine whether this resistance is attributable to the viral neuraminidase, we used a cloned variant HPF3 HN that has two amino acid alterations in HN leading to the loss of detectable neuraminidase activity. Cells expressing the neuraminidase-deficient variant HN-GFP were not protected from infection, despite expressing HN on their surface at levels even higher than the wild-type cell clones. Our results demonstrate that the HPF3 HN-mediated interference effect can be attributed to the presence of an active neuraminidase enzyme activity and provide the first definitive evidence that the mechanism for attachment interference by a paramyxovirus is attributable to the viral neuraminidase.

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Breakthrough of SIV strain smE660 challenge in SIV strain mac239-vaccinated rhesus macaques despite potent autologous neutralizing antibody responses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1509731112 ◽

2015 ◽

Vol 112 (34) ◽

pp. 10780-10785 ◽

Cited By ~ 21

Author(s):

Samantha L. Burton ◽

Katie M. Kilgore ◽

S. Abigail Smith ◽

Sharmila Reddy ◽

Eric Hunter ◽

...

Keyword(s):

Simian Immunodeficiency Virus ◽

Neutralizing Antibodies ◽

Rhesus Macaques ◽

Neutralizing Antibody ◽

Active Immunization ◽

Antibody Responses ◽

Challenge Virus ◽

Gm Csf ◽

Immunodeficiency Virus

Although the correlates of immunological protection from human immunodeficiency virus or simian immunodeficiency virus infection remain incompletely understood, it is generally believed that medium to high titers of serum neutralizing antibodies (nAbs) against the challenge virus will prevent infection. This paradigm is based on a series of studies in which passive transfer of HIV-specific nAbs protected rhesus macaques (RMs) from subsequent mucosal challenge with a chimeric human/simian immunodeficiency virus. However, it is unknown whether nAb titers define protection in the setting of active immunization. Here we determined serum nAb titers against breakthrough transmitted/founder (T/F) SIVsmE660-derived envelope glycoprotein (Env) variants from 14 RMs immunized with SIVmac239-based DNA-prime/modified vaccinia virus Ankara-boost vaccine regimens that included GM-CSF or CD40L adjuvants and conferred significant but incomplete protection against repeated low-dose intrarectal challenge. A single Env variant established infection in all RMs except one, with no identifiable genetic signature associated with vaccination breakthrough compared with T/F Envs from four unvaccinated monkeys. Breakthrough T/F Env pseudoviruses were potently neutralized in vitro by heterologous pooled serum from chronically SIVsmE660-infected monkeys at IC50 titers exceeding 1:1,000,000. Remarkably, the T/F Env pseudoviruses from 13 of 14 monkeys were also susceptible to neutralization by autologous prechallenge serum at in vitro IC50 titers ranging from 1:742–1:10,832. These titers were similar to those observed in vaccinated RMs that remained uninfected. These data suggest that the relationship between serum nAb titers and protection from mucosal SIV challenge in the setting of active immunization is more complex than previously recognized, warranting further studies into the balance between immune activation, target cell availability, and protective antibody responses.

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Specific Phosphorylated Forms of Glyceraldehyde 3-Phosphate Dehydrogenase Associate with Human Parainfluenza Virus Type 3 and Inhibit Viral Transcription In Vitro

Journal of Virology ◽

10.1128/jvi.74.8.3634-3641.2000 ◽

2000 ◽

Vol 74 (8) ◽

pp. 3634-3641 ◽

Cited By ~ 20

Author(s):

Suresh Choudhary ◽

Bishnu P. De ◽

Amiya K. Banerjee

Keyword(s):

Molecular Species ◽

Cell Extract ◽

Parainfluenza Virus ◽

Viral Transcription ◽

Cytoplasmic Fraction ◽

La Protein ◽

Transcription In Vitro ◽

Human Parainfluenza Virus ◽

Type 3

ABSTRACT We previously reported specific interaction of cellular glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the key glycolytic enzyme, and La protein, the RNA polymerase III transcription factor, with the cis-acting RNAs of human parainfluenza virus type 3 (HPIV3) and packaging of these proteins within purified virions (B. P. De, S. Gupta, H. Zhao, J. Z. Drazba, and A. K. Banerjee, J. Biol. Chem. 271:24728–24735, 1996). To gain further insight into these molecular interactions, we analyzed the virion-associated GAPDH and La protein using two-dimensional gel electrophoresis and immunoblotting. The GAPDH was resolved into two major and one minor molecular species migrating in the pI range of 7.6 to 8.3, while the La protein was resolved into five molecular species in the pI range of 6.8 to 7.5. The GAPDH isoforms present in the virions were also detected in the cytoplasmic fraction of CV-1 cell extract, albeit as minor species. On the other hand, the multiple molecular forms of La protein as seen within the virions were readily detected in the total CV-1 cell extract. Further analysis of virion-associated GAPDH by in vivo labeling with [32P]orthophosphate revealed the presence of multiple phosphorylated species. The phosphorylated species were able to bind specifically to the viral cis-acting 3′ genome sense RNA but failed to bind to the leader sense RNA, as determined by gel mobility shift assay. In contrast, the La protein isoforms present within the virions were not phosphorylated and bound to the viralcis-acting RNAs in a phosphorylation-independent manner. The GAPDH isoforms purified from the CV-1 cell cytoplasmic fraction inhibited viral transcription in vitro. Consistent with this, flag-tagged recombinant GAPDH synthesized by using the vaccinia virus expression system also inhibited viral transcription. Together, these data indicate that specific phosphorylated forms of GAPDH associate with HPIV3 and are involved in the regulation of virus gene expression.

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Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector

Journal of Virology ◽

10.1128/jvi.01196-16 ◽

2016 ◽

Vol 90 (21) ◽

pp. 10022-10038 ◽

Cited By ~ 21

Author(s):

Bo Liang ◽

Joan O. Ngwuta ◽

Richard Herbert ◽

Joanna Swerczek ◽

David W. Dorward ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Rhesus Monkeys ◽

Neutralizing Antibodies ◽

Vaccine Candidate ◽

Parainfluenza Virus ◽

F Protein ◽

Syncytial Virus ◽

Human Parainfluenza Virus ◽

Type 3

ABSTRACTHuman respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major pediatric respiratory pathogens that lack vaccines. A chimeric bovine/human PIV3 (rB/HPIV3) virus expressing the unmodified, wild-type (wt) RSV fusion (F) protein from an added gene was previously evaluated in seronegative children as a bivalent intranasal RSV/HPIV3 vaccine, and it was well tolerated but insufficiently immunogenic for RSV F. We recently showed that rB/HPIV3 expressing a partially stabilized prefusion form (pre-F) of RSV F efficiently induced “high-quality” RSV-neutralizing antibodies, defined as antibodies that neutralize RSVin vitrowithout added complement (B. Liang et al., J Virol 89:9499–9510, 2015, doi:10.1128/JVI.01373-15). In the present study, we modified RSV F by replacing its cytoplasmic tail (CT) domain or its CT and transmembrane (TM) domains (TMCT) with counterparts from BPIV3 F, with or without pre-F stabilization. This resulted in RSV F being packaged in the rB/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10- to 100-fold) and rhesus monkeys (100- to 1,000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of high-quality RSV-neutralizing serum antibodies in hamsters. In rhesus monkeys, a strongly additive immunogenic effect of packaging and pre-F stabilization was observed, as demonstrated by 8- and 30-fold increases of RSV-neutralizing serum antibody titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone. Analysis of vaccine-induced F-specific antibodies by binding assays indicated that packaging conferred substantial stabilization of RSV F in the pre-F conformation. This provides an improved version of this well-tolerated RSV/HPIV3 vaccine candidate, with potently improved immunogenicity, which can be returned to clinical trials.IMPORTANCEHuman respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major viral agents of acute pediatric bronchiolitis and pneumonia worldwide that lack vaccines. A bivalent intranasal RSV/HPIV3 vaccine candidate consisting of a chimeric bovine/human PIV3 (rB/HPIV3) strain expressing the RSV fusion (F) protein was previously shown to be well tolerated by seronegative children but was insufficiently immunogenic for RSV F. In the present study, the RSV F protein was engineered to be packaged efficiently into vaccine virus particles. This resulted in a significantly enhanced quantity and quality of RSV-neutralizing antibodies in hamsters and nonhuman primates. In nonhuman primates, this effect was strongly additive to the previously described stabilization of the prefusion conformation of the F protein. The improved immunogenicity of RSV F by packaging appeared to involve prefusion stabilization. These findings provide a potently more immunogenic version of this well-tolerated vaccine candidate and should be applicable to other vectored vaccines.

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Generation of a Candidate Live Marker Vaccine for Equine Arteritis Virus by Deletion of the Major Virus Neutralization Domain

Journal of Virology ◽

10.1128/jvi.77.15.8470-8480.2003 ◽

2003 ◽

Vol 77 (15) ◽

pp. 8470-8480 ◽

Cited By ~ 25

Author(s):

Javier Castillo-Olivares ◽

Roeland Wieringa ◽

Tamás Bakonyi ◽

Antoine A. F. de Vries ◽

Nick J. Davis-Poynter ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Rna Virus ◽

Viral Envelope ◽

Equine Arteritis Virus ◽

Wild Type Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Wild Type ◽

Challenge Virus ◽

Marker Vaccine

ABSTRACT Equine arteritis virus (EAV) is an enveloped plus-strand RNA virus of the family Arteriviridae (order Nidovirales) that causes respiratory and reproductive disease in equids. Protective, virus-neutralizing antibodies (VNAb) elicited by infection are directed predominantly against an immunodominant region in the membrane-proximal domain of the viral envelope glycoprotein GL, allowing recently the establishment of a sensitive peptide enzyme-linked immunosorbent assay (ELISA) based on this particular domain (J. Nugent et al., J. Virol. Methods 90:167-183, 2000). By using an infectious cDNA we have now generated, in the controlled background of a nonvirulent virus, a mutant EAV from which this immunodominant domain was deleted. This virus, EAV-GLΔ, replicated to normal titers in culture cells, although at a slower rate than wild-type EAV, and caused an asymptomatic infection in ponies. The antibodies induced neutralized the mutant virus efficiently in vitro but reacted poorly to wild-type EAV strains. Nevertheless, when inoculated subsequently with virulent EAV, the immunized animals, in contrast to nonvaccinated controls, were fully protected against disease; replication of the challenge virus occurred briefly at low though detectable levels. The levels of protection achieved suggest that an immune effector mechanism other than VNAb plays an important role in protection against infection. As expected, infection with EAV-GLΔ did not induce a measurable response in our GL-peptide ELISA while the challenge infection of the animals clearly did. EAV-GLΔ or similar mutants are therefore attractive marker vaccine candidates, enabling serological discrimination between vaccinated and wild-type virus-infected animals.

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Role of Env in Resistance of Feline Immunodeficiency Virus (FIV)-Infected Cats to Superinfection by a Second FIV Strain as Determined by Using a Chimeric Virus

Journal of Virology ◽

10.1128/jvi.01064-07 ◽

2007 ◽

Vol 81 (19) ◽

pp. 10474-10485 ◽

Cited By ~ 4

Author(s):

Simone Giannecchini ◽

Mauro Pistello ◽

Patrizia Isola ◽

Donatella Matteucci ◽

Paola Mazzetti ◽

...

Keyword(s):

Feline Immunodeficiency Virus ◽

Neutralizing Antibodies ◽

Viral Envelope ◽

Lymphoid Cells ◽

Time Interval ◽

Complete Protection ◽

Challenge Virus ◽

Immunodeficiency Virus

ABSTRACT A more or less pronounced resistance to superinfection by a second strain of the infecting virus has been observed in many lentivirus-infected hosts. We used a chimeric feline immunodeficiency virus (FIV), designated FIVχ, containing a large part of the env gene of a clade B virus (strain M2) and all the rest of the genome of a clade A virus (a p34TF10 molecular clone of the Petaluma strain modified to grow in lymphoid cells), to gain insights into such resistance. FIVχ was infectious and moderately pathogenic for cats and in vitro exhibited the neutralization specificity of the env donor. The experiments performed were bidirectional, in that cats preinfected with either parental virus were challenged with FIVχ and vice versa. The preinfected animals were partially or completely protected relative to what was observed in naïve control animals, most likely due, at least in part, to the circumstance that in all the preinfecting/challenge virus combinations examined, the first and the second virus shared significant viral components. Based on the proportions of complete protection observed, the role of a strongly matched viral envelope appeared to be modest and possibly dependent on the time interval between the first and the second infection. Furthermore, complete protection and the presence of measurable neutralizing antibodies capable of blocking the second virus in vitro were not associated.

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