Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models

ABSTRACTZika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) werede novosynthesized and recovered by reverse genetics and contained large amounts of underrepresented codon pairs in the E gene and/or NS1 gene. The amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ≫ Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections.IMPORTANCEDue to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms, including Guillain-Barré syndrome, microcephaly, and other birth defects in humans, there is an urgent need for ZIKV vaccine development. Here we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammal attenuated and preferred insect to mammalian cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible for the variant to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live attenuated vaccine candidates against flaviviruses such as ZIKV, Japanese encephalitis virus, and West Nile virus.

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Vaccine candidates generated by codon and codon pair deoptimization of enterovirus A71 protect against lethal challenge in mice

Vaccine ◽

10.1016/j.vaccine.2021.02.024 ◽

2021 ◽

Vol 39 (12) ◽

pp. 1708-1720

Author(s):

Michelle Hui Pheng Lee ◽

Chee Wah Tan ◽

Han Kang Tee ◽

Kien Chai Ong ◽

I-Ching Sam ◽

...

Keyword(s):

Lethal Challenge ◽

Vaccine Candidates ◽

Enterovirus A71 ◽

Codon Pair

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2764. Generation of a Balanced, Tetravalent Dengue Vaccine Based on Contemporary Strains Using a Computational, Synthetic Biology-Based Platform

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.2441 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S975-S975

Author(s):

Ying Wang ◽

Charles B Stauft ◽

Kanakatte Raviprakash ◽

J Robert Coleman ◽

Steffen Mueller

Keyword(s):

Human Cell ◽

Neutralizing Antibodies ◽

Vaccine Dose ◽

Neutralizing Antibody ◽

Denv Infection ◽

The United States ◽

Wild Type ◽

Lethal Challenge ◽

Denv Serotypes ◽

Tetravalent Vaccine

Abstract Background The WHO estimates that there may be 50 million cases of dengue virus (DENV) infection worldwide every year. There is no safe vaccine against DENV licensed in the United States. The development of a balanced and effective anti-DENV vaccine is vital to preventing morbidity and mortality. Codagenix used its proprietary SAVE (Synthetic Attenuated Virus Engineering) platform to generate and test a live attenuated, tetravalent vaccine against DENV. Methods Codagenix used SAVE to substitute under-represented human codons and codon-pairs into the E protein sequences of contemporary strains of DENV1-4, producing either a fully human-cell-deoptimized prM-E (E-Min), or a partially deoptimized prM-E (E-W/Min) to allow for balancing of the vaccine’s immunogenicity. Full genomes containing deoptimized E-Min and E-W/Min in the DENV2 backbone were transfected into cells to recover live-attenuated, human-cell-deoptimized vaccine strains. Mice were vaccinated with 106 FFU of each DENV vaccine (alone or together), boosted on day 21 and assessed for neutralizing antibodies by PRNT50 and survival after lethal challenge with mouse-adapted wild-type (WT) DENV. Cynomolgus macaques were immunized with a mixture of 106 FFU of each DENV vaccine strain. Two doses were administered on study day 1 and 57 and serum neutralizing antibodies were determined on day 57 and 85 by a microneutralization assay. Results SAVE deoptimized DENV viruses grew to wild-type (between 107 and 108 FFU/ml) levels at permissive temperatures (<37C). All vaccine strains generated neutralizing antibody levels comparable to WT. A tetravalent formulation containing all four E-Min strains protected mice from lethal challenge with DENV3. A tetravalent formulation of Codagenix DENV-E-W/Min vaccine elicited a robust and balanced neutralizing antibody response in non-human primates (NHPs) against all four DENV serotypes after a single dose. A second vaccine dose did not boost antibody titers significantly. Conclusion The ability to rationally balance the attenuation of multiple vaccine strains, thereby avoiding antibody-dependent enhancement, is a unique advantage of the Codagenix SAVE platform. Codagenix DENV vaccine viruses generated balanced, sterilizing immunity in NHPs after one dose. Disclosures All authors: No reported disclosures.

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Protective efficacy of rhesus adenovirus COVID-19 vaccines against mouse-adapted SARS-CoV-2

Journal of Virology ◽

10.1128/jvi.00974-21 ◽

2021 ◽

Author(s):

Lisa H. Tostanoski ◽

Lisa E. Gralinski ◽

David R. Martinez ◽

Alexandra Schaefer ◽

Shant H. Mahrokhian ◽

...

Keyword(s):

Viral Replication ◽

Protective Efficacy ◽

Neutralizing Antibody ◽

Wild Type ◽

Adenovirus Serotype ◽

Vaccine Candidates ◽

Correlates Of Protection ◽

Immune Correlates ◽

Antibody Titers ◽

Adenovirus Vectors

The global COVID-19 pandemic has sparked intense interest in the rapid development of vaccines as well as animal models to evaluate vaccine candidates and to define immune correlates of protection. We recently reported a mouse-adapted SARS-CoV-2 virus strain (MA10) with the potential to infect wild-type laboratory mice, driving high levels of viral replication in respiratory tract tissues as well as severe clinical and respiratory symptoms, aspects of COVID-19 disease in humans that are important to capture in model systems. We evaluated the immunogenicity and protective efficacy of novel rhesus adenovirus serotype 52 (RhAd52) vaccines against MA10 challenge in mice. Baseline seroprevalence is lower for rhesus adenovirus vectors than for human or chimpanzee adenovirus vectors, making these vectors attractive candidates for vaccine development. We observed that RhAd52 vaccines elicited robust binding and neutralizing antibody titers, which inversely correlated with viral replication after challenge. These data support the development of RhAd52 vaccines and the use of the MA10 challenge virus to screen novel vaccine candidates and to study the immunologic mechanisms that underscore protection from SARS-CoV-2 challenge in wild-type mice. Importance We have developed a series of SARS-CoV-2 vaccines using rhesus adenovirus serotype 52 (RhAd52) vectors, which exhibits a lower seroprevalence than human and chimpanzee vectors, supporting their development as novel vaccine vectors or as an alternative Ad vector for boosting. We sought to test these vaccines using a recently reported mouse-adapted SARS-CoV-2 (MA10) virus to i) evaluate the protective efficacy of RhAd52 vaccines and ii) further characterize this mouse-adapted challenge model and probe immune correlates of protection. We demonstrate RhAd52 vaccines elicit robust SARS-CoV-2-specific antibody responses and protect against clinical disease and viral replication in the lungs. Further, binding and neutralizing antibody titers correlated with protective efficacy. These data validate the MA10 mouse model as a useful tool to screen and study novel vaccine candidates, as well as the development of RhAd52 vaccines for COVID-19.

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An Attenuated Zika Virus Encoding Non-Glycosylated Envelope (E) and Non-Structural Protein 1 (NS1) Confers Complete Protection against Lethal Challenge in a Mouse Model

Vaccines ◽

10.3390/vaccines7030112 ◽

2019 ◽

Vol 7 (3) ◽

pp. 112 ◽

Cited By ~ 4

Author(s):

Arun S. Annamalai ◽

Aryamav Pattnaik ◽

Bikash R. Sahoo ◽

Zack P. Guinn ◽

Brianna L. Bullard ◽

...

Keyword(s):

Zika Virus ◽

Structural Protein ◽

Complete Protection ◽

Lethal Challenge ◽

Live Virus ◽

Virus Challenge ◽

Glycosylation Sites ◽

Viral Vaccine ◽

Asian Lineage ◽

Congenital Microcephaly

Zika virus (ZIKV), a mosquito-transmitted flavivirus, emerged in the last decade causing serious human diseases, including congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Although many vaccine platforms are at various stages of development, no licensed vaccines are currently available. Previously, we described a mutant MR766 ZIKV (m2MR) bearing an E protein mutation (N154A) that prevented its glycosylation, resulting in attenuation and defective neuroinvasion. To further attenuate m2MR for its potential use as a live viral vaccine, we incorporated additional mutations into m2MR by substituting the asparagine residues in the glycosylation sites (N130 and N207) of NS1 with alanine residues. Examination of pathogenic properties revealed that the virus (m5MR) carrying mutations in E (N154A) and NS1 (N130A and N207A) was fully attenuated with no disease signs in infected mice, inducing high levels of humoral and cell-mediated immune responses, and protecting mice from subsequent lethal virus challenge. Furthermore, passive transfer of sera from m5MR-infected mice into naïve animals resulted in complete protection from lethal challenge. The immune sera from m5MR-infected animals neutralized both African and Asian lineage viruses equally well, suggesting that m5MR virus could be developed as a potentially broad live virus vaccine candidate.

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Search for Correlates of Protective Immunity Conferred by Anthrax Vaccine

Infection and Immunity ◽

10.1128/iai.69.5.2888-2893.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 2888-2893 ◽

Cited By ~ 175

Author(s):

Shaul Reuveny ◽

Moshe D. White ◽

Yaakov Y. Adar ◽

Yaron Kafri ◽

Zeev Altboum ◽

...

Keyword(s):

Antibody Titer ◽

Neutralizing Antibodies ◽

Protective Immunity ◽

Protective Antigen ◽

Surrogate Marker ◽

Neutralizing Antibody ◽

Enzyme Linked Immunosorbent Assay ◽

Lethal Challenge ◽

Anthrax Spores ◽

Antibody Titers

ABSTRACT Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (r 2 = 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD50) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.

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Development of a potent Zika virus vaccine using self-amplifying messenger RNA

Science Advances ◽

10.1126/sciadv.aba5068 ◽

2020 ◽

Vol 6 (32) ◽

pp. eaba5068 ◽

Cited By ~ 1

Author(s):

Kate Luisi ◽

Kaitlyn M. Morabito ◽

Katherine E. Burgomaster ◽

Mayuri Sharma ◽

Wing-Pui Kong ◽

...

Keyword(s):

Zika Virus ◽

Messenger Rna ◽

Protective Immunity ◽

Nonhuman Primates ◽

Neutralizing Antibody ◽

Effective Vaccine ◽

Proof Of Concept ◽

Zikv Infection ◽

Vaccine Candidates ◽

Rapid Responses

Zika virus (ZIKV) is the cause of a pandemic associated with microcephaly in newborns and Guillain-Barre syndrome in adults. Currently, there are no available treatments or vaccines for ZIKV, and the development of a safe and effective vaccine is a high priority for many global health organizations. We describe the development of ZIKV vaccine candidates using the self-amplifying messenger RNA (SAM) platform technology delivered by cationic nanoemulsion (CNE) that allows bedside mixing and is particularly useful for rapid responses to pandemic outbreaks. Two immunizations of either of the two lead SAM (CNE) vaccine candidates elicited potent neutralizing antibody responses to ZIKV in mice and nonhuman primates. Both SAM (CNE) vaccines protected these animals from ZIKV challenge, with one candidate providing complete protection against ZIKV infection in nonhuman primates. The data provide a preclinical proof of concept that a SAM (CNE) vaccine candidate can rapidly elicit protective immunity against ZIKV.

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Characterization of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein

Nature Communications ◽

10.1038/s41467-021-23166-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Pui Wang ◽

Siu-Ying Lau ◽

Shaofeng Deng ◽

Pin Chen ◽

Bobo Wing-Yee Mok ◽

...

Keyword(s):

Neutralizing Antibody ◽

T Cell Response ◽

Spike Protein ◽

Wild Type Virus ◽

Wild Type ◽

Cleavage Motif ◽

Sterilizing Immunity

AbstractSARS-CoV-2 is of zoonotic origin and contains a PRRA polybasic cleavage motif which is considered critical for efficient infection and transmission in humans. We previously reported on a panel of attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction of the spike protein. Here, we characterize pathogenicity, immunogenicity, and protective ability of a further cell-adapted SARS-CoV-2 variant, Ca-DelMut, in in vitro and in vivo systems. Ca-DelMut replicates more efficiently than wild type or parental virus in Vero E6 cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut causes no obvious pathological changes and does not induce elevation of proinflammatory cytokines, but still triggers a strong neutralizing antibody and T cell response in hamsters and mice. Ca-DelMut immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, with little sign of virus replication in the upper or lower respiratory tract, demonstrating sterilizing immunity.

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Distinct neutralizing antibody correlates of protection among related Zika virus vaccines identify a role for antibody quality

Science Translational Medicine ◽

10.1126/scitranslmed.aaw9066 ◽

2020 ◽

Vol 12 (547) ◽

pp. eaaw9066

Author(s):

Sonia Maciejewski ◽

Tracy J. Ruckwardt ◽

Kaitlyn M. Morabito ◽

Bryant M. Foreman ◽

Katherine E. Burgomaster ◽

...

Keyword(s):

Zika Virus ◽

Nonhuman Primates ◽

Neutralizing Antibody ◽

Structural Proteins ◽

Zikv Infection ◽

Vaccine Candidates ◽

Correlates Of Protection ◽

Vaccine Antigens ◽

Maturation State ◽

Induced Immunity

The emergence of Zika virus (ZIKV) in the Americas stimulated the development of multiple ZIKV vaccine candidates. We previously developed two related DNA vaccine candidates encoding ZIKV structural proteins that were immunogenic in animal models and humans. We sought to identify neutralizing antibody (NAb) properties induced by each vaccine that correlated with protection in nonhuman primates (NHPs). Despite eliciting equivalent NAb titers in NHPs, these vaccines were not equally protective. The transfer of equivalent titers of vaccine-elicited NAb into AG129 mice also revealed nonequivalent protection, indicating qualitative differences among antibodies (Abs) elicited by these vaccines. Both vaccines elicited Abs with similar binding titers against envelope protein monomers and those incorporated into virus-like particles, as well as a comparable capacity to orchestrate phagocytosis. Functional analysis of vaccine-elicited NAbs from NHPs and humans revealed a capacity to neutralize the structurally mature form of the ZIKV virion that varied in magnitude among vaccine candidates. Conversely, sensitivity to the virion maturation state was not a characteristic of NAbs induced by natural or experimental infection. Passive transfer experiments in mice revealed that neutralization of mature ZIKV virions more accurately predicts protection from ZIKV infection. These findings demonstrate that NAb correlates of protection may differ among vaccine antigens when assayed using standard neutralization platforms and suggest that measurements of Ab quality, including the capacity to neutralize mature virions, will be critical for defining correlates of ZIKV vaccine-induced immunity.

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Pathogenicity, immunogenicity, and protective ability of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein

10.21203/rs.3.rs-78635/v1 ◽

2020 ◽

Author(s):

Pui Wang ◽

Siu-Ying Lau ◽

Shaofeng Deng ◽

Pin Chen ◽

Bobo Mok ◽

...

Keyword(s):

Lower Respiratory Tract ◽

Neutralizing Antibody ◽

Spike Protein ◽

Wild Type Virus ◽

Wild Type ◽

Protective Ability ◽

Cleavage Motif ◽

Sterilizing Immunity

Abstract SARS-CoV-2 is zoonotic origin and contains a PRRA polybasic cleavage motif which is considered critical for efficient infection and transmission in humans. We previously reported on a panel of attenuated SARS-CoV-2 variants with deletion at the S1/S2 junction of spike protein. Here we characterize pathogenicity, immunogenicity, and protective ability of a further cell-adapted SARS-CoV-2 variant, Ca-DelMut, in in vitro and in vivo systems. Ca-DelMut replicates more efficiently than wild type or parental virus in cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut causes no apparent pathological changes and does not induce elevated proinflammatory cytokines in hamster infections, but still triggers a strong neutralizing antibody response in hamsters. Ca-DelMut immunized hamsters challenged with wild type SARS-CoV-2 are fully protected with no sign of virus replication in the upper or lower respiratory tract of challenged animals, demonstrating sterilizing immunity.

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Pathogenicity, immunogenicity, and protective ability of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein

10.1101/2020.08.24.264192 ◽

2020 ◽

Author(s):

Pui Wang ◽

Siu-Ying Lau ◽

Shaofeng Deng ◽

Pin Chen ◽

Bobo Wing-Yee Mok ◽

...

Keyword(s):

Antibody Response ◽

Proinflammatory Cytokines ◽

Neutralizing Antibody ◽

Spike Protein ◽

Parental Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Cleavage Motif ◽

Sterilizing Immunity

AbstractSARS-CoV-2 contains a PRRA polybasic cleavage motif considered critical for efficient infection and transmission in humans. We previously reported that virus variants with spike protein S1/S2 junction deletions spanning this motif are attenuated. Here we characterize a further cell-adapted SARS-CoV-2 variant, Ca-DelMut. Ca-DelMut replicates more efficiently than wild type or parental virus in cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut does not induce proinflammatory cytokines in hamster infections, but still triggers a strong neutralizing antibody response. Ca-DelMut-immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, demonstrating sterilizing immunity.

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