Chikungunya Virus Vaccine Candidates with Decreased Mutational Robustness Are Attenuated In Vivo and Have Compromised Transmissibility

ABSTRACT Chikungunya virus (CHIKV) is a reemerged arbovirus, a member of the Togaviridae family. It circulates through mosquito vectors mainly of the Aedes family and a mammalian host. CHIKV causes chikungunya fever, a mild to severe disease characterized by arthralgia, with some fatal outcomes described. In the past years, several outbreaks mainly caused by enhanced adaptation of the virus to the vector and ineffective control of the contacts between infected mosquito populations and the human host have been reported. Vaccines represent the best solution for the control of insect-borne viruses, including CHIKV, but are often unavailable. We designed live attenuated CHIKVs by applying a rational genomic design based on multiple replacements of synonymous codons. In doing so, the virus mutational robustness (capacity to maintain phenotype despite introduction of mutations to genotype) is decreased, driving the viral population toward deleterious evolutionary trajectories. When the candidate viruses were tested in the insect and mammalian hosts, we observed overall strong attenuation in both and greatly diminished signs of disease. Moreover, we found that the vaccine candidates elicited protective immunity related to the production of neutralizing antibodies after a single dose. During an experimental transmission cycle between mosquitoes and naive mice, vaccine candidates could be transmitted by mosquito bite, leading to asymptomatic infection in mice with compromised dissemination. Using deep-sequencing technology, we observed an increase in detrimental (stop) codons, which confirmed the effectiveness of this genomic design. Because the approach involves hundreds of synonymous modifications to the genome, the reversion risk is significantly reduced, rendering the viruses promising vaccine candidates. IMPORTANCE Chikungunya fever is a debilitating disease that causes severe pain to the joints, which can compromise the patient’s lifestyle for several months and even in some grave cases lead to death. The etiological agent is chikungunya virus, an alphavirus transmitted by mosquito bite. Currently, there are no approved vaccines or treatments against the disease. In our research, we developed novel live attenuated vaccine candidates against chikungunya virus by applying an innovative genomic design. When tested in the insect and mammalian host, the vaccine candidates did not cause disease, elicited strong protection against further infection, and had low risk of reversion to pathogenic phenotypes.

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In contrast to TH2-biased approaches, TH1 COVID-19 vaccines protect Syrian hamsters from severe disease in the absence of dexamethasone-treatable vaccine-associated enhanced respiratory pathology

10.1101/2021.12.28.474359 ◽

2021 ◽

Author(s):

Aileen Ebenig ◽

Samada Muraleedharan ◽

Julia Kazmierski ◽

Daniel Todt ◽

Arne Auste ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Viral Vectors ◽

Severe Disease ◽

Lymphoid Cells ◽

Protein Vaccine ◽

S Protein ◽

Vaccine Candidates ◽

Syrian Hamsters ◽

Unvaccinated Control ◽

Low Passage

Since December 2019, the novel human coronavirus SARS-CoV-2 has spread globally, causing millions of deaths. Unprecedented efforts have enabled development and authorization of a range of vaccines, which reduce transmission rates and confer protection against the associated disease COVID-19. These vaccines are conceptually diverse, including e.g. classical adjuvanted whole-inactivated virus, viral vectors, and mRNA vaccines. We have analysed two prototypic model vaccines, the strongly TH1-biased measles vaccine-derived candidate MeVvac2-SARS2-S(H) and a TH2-biased Alum-adjuvanted, non-stabilized Spike (S) protein side-by-side, for their ability to protect Syrian hamsters upon challenge with a low-passage SARS-CoV-2 patient isolate. As expected, the MeVvac2-SARS2-S(H) vaccine protected the hamsters safely from severe disease. In contrast, the protein vaccine induced vaccine-associated enhanced respiratory disease (VAERD) with massive infiltration of eosinophils into the lungs. Global RNA-Seq analysis of hamster lungs revealed reduced viral RNA and less host dysregulation in MeVvac2-SARS2-S(H) vaccinated animals, while S protein vaccination triggered enhanced host gene dysregulation compared to unvaccinated control animals. Of note, mRNAs encoding the major eosinophil attractant CCL-11, the TH2 response-driving cytokine IL-19, as well as TH2-cytokines IL-4, IL-5, and IL-13 were exclusively up-regulated in the lungs of S protein vaccinated animals, consistent with previously described VAERD induced by RSV vaccine candidates. IL-4, IL-5, and IL-13 were also up-regulated in S-specific splenocytes after protein vaccination. Using scRNA-Seq, T cells and innate lymphoid cells were identified as the source of these cytokines, while Ccl11 and Il19 mRNAs were expressed in lung macrophages displaying an activated phenotype. Interestingly, the amount of viral reads in this macrophage population correlated with the abundance of Fc-receptor reads. These findings suggest that VAERD is triggered by induction of TH2-type helper cells secreting IL-4, IL-5, and IL-13, together with stimulation of macrophage subsets dependent on non-neutralizing antibodies. Via this mechanism, uncontrolled eosinophil recruitment to the infected tissue occurs, a hallmark of VAERD immunopathogenesis. These effects could effectively be treated using dexamethasone and were not observed in animals vaccinated with MeVvac2-SARS2-S(H). Taken together, our data validate the potential of TH2-biased COVID-19 vaccines and identify the transcriptional mediators that underlie VAERD, but confirm safety of TH1-biased vaccine concepts such as vector-based or mRNA vaccines. Dexamethasone, which is already in use for treatment of severe COVID-19, may alleviate such VAERD, but in-depth scrutiny of any next-generation protein-based vaccine candidates is required, prior and after their regulatory approval.

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Atypical Chikungunya virus infections: clinical manifestations, mortality and risk factors for severe disease during the 2005–2006 outbreak on Réunion

Epidemiology and Infection ◽

10.1017/s0950268808001167 ◽

2008 ◽

Vol 137 (4) ◽

pp. 534-541 ◽

Cited By ~ 299

Author(s):

A. ECONOMOPOULOU ◽

M. DOMINGUEZ ◽

B. HELYNCK ◽

D. SISSOKO ◽

O. WICHMANN ◽

...

Keyword(s):

Risk Factors ◽

Chikungunya Virus ◽

Viral Infections ◽

Case Reports ◽

Severe Disease ◽

Clinical Manifestations ◽

Chikungunya Fever ◽

Course Of Illness ◽

Atypical Case ◽

Incidence And Mortality

SUMMARYIn April 2005, an outbreak of Chikungunya fever occurred on the island of Réunion in the Indian Ocean. During winter 2005, six patients developed meningoencephalitis and acute hepatitis due to Chikungunya virus. Our objectives were to determine the incidence and mortality of atypical Chikungunya viral infections and to identify risk factors for severe disease. A hospital-based surveillance system was established to collect data on atypical Chikungunya cases. Case reports, medical records and laboratory results were reviewed and analysed. We defined an atypical case as one in which a patient with laboratory-confirmed Chikungunya virus infection developed symptoms other than fever and arthralgia. We defined a severe atypical case as one which required maintenance of at least one vital function. We recorded 610 atypical cases of Chikungunya fever: 222 were severe cases, 65 affected patients died. Five hundred and forty-six cases had underlying medical conditions (of which 226 suffered from cardiovascular, 147 from neurological and 150 from respiratory disorders). Clinical features that had never been associated with Chikungunya fever were recorded, such as bullous dermatosis, pneumonia, and diabetes mellitus. Hypertension, and underlying respiratory or cardiological conditions were independent risk factors for disease severity. The overall mortality rate was 10·6% and it increased with age. This is the first time that severe cases and deaths due to Chikungunya fever have been documented. The information presented in this article may assist clinicians in identifying the disease, selecting the treatment strategy, and anticipating the course of illness.

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Modified Vaccinia Virus Ankara as a Viral Vector for Vaccine Candidates against Chikungunya Virus

Biomedicines ◽

10.3390/biomedicines9091122 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1122

Author(s):

Juan García-Arriaza ◽

Mariano Esteban ◽

Daniel López

Keyword(s):

Vaccinia Virus ◽

Chikungunya Virus ◽

Vaccine Development ◽

Viral Vector ◽

Severe Disease ◽

Febrile Illness ◽

Structural Proteins ◽

Effective Vaccine ◽

Vaccine Candidates ◽

Modified Vaccinia Virus Ankara

There is a need to develop a highly effective vaccine against the emerging chikungunya virus (CHIKV), a mosquito-borne Alphavirus that causes severe disease in humans consisting of acute febrile illness, followed by chronic debilitating polyarthralgia and polyarthritis. In this review, we provide a brief history of the development of the first poxvirus vaccines that led to smallpox eradication and its implications for further vaccine development. As an example, we summarize the development of vaccine candidates based on the modified vaccinia virus Ankara (MVA) vector expressing different CHIKV structural proteins, paying special attention to MVA-CHIKV expressing all of the CHIKV structural proteins: C, E3, E2, 6K and E1. We review the characterization of innate and adaptive immune responses induced in mice and nonhuman primates by the MVA-CHIKV vaccine candidate and examine its efficacy in animal models, with promising preclinical findings needed prior to the approval of human clinical trials.

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A SARS-CoV-2 vaccine candidate would likely match all currently circulating variants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008281117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23652-23662 ◽

Cited By ~ 25

Author(s):

Bethany Dearlove ◽

Eric Lewitus ◽

Hongjun Bai ◽

Yifan Li ◽

Daniel B. Reeves ◽

...

Keyword(s):

Viral Entry ◽

Neutralizing Antibodies ◽

Vaccine Candidate ◽

Spike Protein ◽

Reference Sequence ◽

Viral Population ◽

Effective Vaccine ◽

Nucleotide Substitutions ◽

Bioinformatics Analyses ◽

Vaccine Candidates

The magnitude of the COVID-19 pandemic underscores the urgency for a safe and effective vaccine. Many vaccine candidates focus on the Spike protein, as it is targeted by neutralizing antibodies and plays a key role in viral entry. Here we investigate the diversity seen in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences and compare it to the sequence on which most vaccine candidates are based. Using 18,514 sequences, we perform phylogenetic, population genetics, and structural bioinformatics analyses. We find limited diversity across SARS-CoV-2 genomes: Only 11 sites show polymorphisms in >5% of sequences; yet two mutations, including the D614G mutation in Spike, have already become consensus. Because SARS-CoV-2 is being transmitted more rapidly than it evolves, the viral population is becoming more homogeneous, with a median of seven nucleotide substitutions between genomes. There is evidence of purifying selection but little evidence of diversifying selection, with substitution rates comparable across structural versus nonstructural genes. Finally, the Wuhan-Hu-1 reference sequence for the Spike protein, which is the basis for different vaccine candidates, matches optimized vaccine inserts, being identical to an ancestral sequence and one mutation away from the consensus. While the rapid spread of the D614G mutation warrants further study, our results indicate that drift and bottleneck events can explain the minimal diversity found among SARS-CoV-2 sequences. These findings suggest that a single vaccine candidate should be efficacious against currently circulating lineages.

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Enhancement of alphavirus replication in mammalian cells at sub-physiological temperatures

10.1101/2021.09.29.462334 ◽

2021 ◽

Author(s):

Jinchao Guo ◽

Mark Harris

Keyword(s):

Mammalian Cells ◽

Chikungunya Virus ◽

Stress Granules ◽

Mammalian Host ◽

Temperature Sensitive ◽

Mosquito Vector ◽

Structural Protein ◽

Physiological Temperature ◽

Mosquito Bite ◽

Mosquito Cells

AbstractChikungunya virus (CHIKV) is a re-emerging Alphavirus transmitted by Aedes mosquitoes and causing fever, rash and arthralgia. Currently there are no vaccines or antiviral agents against CHIKV, therefore it is important to understand the molecular details of CHIKV replication. In this regard, the function of the Alphavirus non-structural protein 3 (nsP3) remains enigmatic. Building on previous studies (Gao et al, 2019), we generated a panel of mutants in a conserved and surface exposed cluster in the nsP3 alphavirus unique domain (AUD) and tested their replication phenotype using a sub-genomic replicon (SGR) in mammalian and mosquito cells. We identified three mutants that replicated poorly in mammalian cells but showed no defect in mosquito cells. We further showed that these mutants were temperature-sensitive, rather than species-specific, as they exhibited no replication defect in mammalian cells at sub-physiological temperature (28°C). Similar effects were observed in the context of infectious CHIKV as well as a closely related virus: O’Nyong Nyong virus (ONNV). Intriguingly, this analysis also revealed that the wildtype SGR replicated much more efficiently at sub-physiological temperature as compared to 37°C. This was not due to impaired interferon responses as this enhancement was also observed in Vero cells. Neither was this due to a defect in the phosphorylation of eIF2α as treatment with ISRIB, an inhibitor of global translation attenuation, did not compensate for replication defects at 37°C. However, we noticed significant differences between the sizes and numbers of virus-induced stress granules (SG) at physiological and sub-physiological temperatures. As cells in the periphery will be at sub-physiological temperatures, and these will be the first cells infected in the mammalian host following a mosquito bite, we propose that alphaviruses have evolved mechanisms to limit antiviral responses in these cells to promote viral genome replication.Author summaryChikungunya virus (CHIKV) is a re-emerging arbovirus. It is transmitted by Aedes species of mosquitos and poses massive epidemic threats. Arboviruses are unique in that they must be able to replicate efficiently in both the mosquito vector and the mammalian host, and therefore at different temperatures. Importantly, the first cells infected in the mammalian host following a mosquito bite will be in the skin and therefore at sub-physiological temperature (lower than 37°C). Here we show that mutants within one of the CHIKV proteins (nsP3) were unable to replicate at 37°C but replicated efficiently in mammalian cells at a sub-physiological temperature (28°C). We also showed that the wildtype virus replicated more efficiently at 28°C in comparison to at 37°C in mammalian cells. We investigated the mechanism behind this phenomenon and showed that at sub-physiological temperatures the virus induced the formation of smaller and more numerous cytoplasmic stress granules. We propose that alphaviruses have evolved mechanisms to promote their replication in mammalian cells at sub-physiological temperatures to facilitate infection of mammals via a cutaneous mosquito bite.

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Protective efficacy of a SARS-CoV-2 DNA vaccine in wild-type and immunosuppressed Syrian hamsters

npj Vaccines ◽

10.1038/s41541-020-00279-z ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Rebecca L. Brocato ◽

Steven A. Kwilas ◽

Robert K. Kim ◽

Xiankun Zeng ◽

Lucia M. Principe ◽

...

Keyword(s):

Dna Vaccine ◽

Neutralizing Antibodies ◽

Protective Efficacy ◽

Phase 1 ◽

Severe Disease ◽

Disease Model ◽

Wild Type ◽

Jet Injection ◽

Syrian Hamsters ◽

Dna Targeting

AbstractA worldwide effort to counter the COVID-19 pandemic has resulted in hundreds of candidate vaccines moving through various stages of research and development, including several vaccines in phase 1, 2 and 3 clinical trials. A relatively small number of these vaccines have been evaluated in SARS-CoV-2 disease models, and fewer in a severe disease model. Here, a SARS-CoV-2 DNA targeting the spike protein and delivered by jet injection, nCoV-S(JET), elicited neutralizing antibodies in hamsters and was protective in both wild-type and transiently immunosuppressed hamster models. This study highlights the DNA vaccine, nCoV-S(JET), we developed has a great potential to move to next stage of preclinical studies, and it also demonstrates that the transiently-immunosuppressed Syrian hamsters, which recapitulate severe and prolonged COVID-19 disease, can be used for preclinical evaluation of the protective efficacy of spike-based COVID-19 vaccines.

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SARS-CoV-2 vaccines strategies: a comprehensive review of phase 3 candidates

npj Vaccines ◽

10.1038/s41541-021-00292-w ◽

2021 ◽

Vol 6 (1) ◽

Cited By ~ 3

Author(s):

Nikolaos C. Kyriakidis ◽

Andrés López-Cortés ◽

Eduardo Vásconez González ◽

Alejandra Barreto Grimaldos ◽

Esteban Ortiz Prado

Keyword(s):

Neutralizing Antibodies ◽

Large Scale ◽

Vaccine Development ◽

Rna Virus ◽

Protein S ◽

Effective Vaccine ◽

Phase 3 ◽

Vaccine Candidates ◽

Advantages And Disadvantages ◽

The World

AbstractThe new SARS-CoV-2 virus is an RNA virus that belongs to the Coronaviridae family and causes COVID-19 disease. The newly sequenced virus appears to originate in China and rapidly spread throughout the world, becoming a pandemic that, until January 5th, 2021, has caused more than 1,866,000 deaths. Hence, laboratories worldwide are developing an effective vaccine against this disease, which will be essential to reduce morbidity and mortality. Currently, there more than 64 vaccine candidates, most of them aiming to induce neutralizing antibodies against the spike protein (S). These antibodies will prevent uptake through the human ACE-2 receptor, thereby limiting viral entrance. Different vaccine platforms are being used for vaccine development, each one presenting several advantages and disadvantages. Thus far, thirteen vaccine candidates are being tested in Phase 3 clinical trials; therefore, it is closer to receiving approval or authorization for large-scale immunizations.

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Neutralizing Antibody Therapeutics for COVID-19

Viruses ◽

10.3390/v13040628 ◽

2021 ◽

Vol 13 (4) ◽

pp. 628

Author(s):

Aeron C. Hurt ◽

Adam K. Wheatley

Keyword(s):

High Risk ◽

Neutralizing Antibodies ◽

Controlled Trial ◽

Antiviral Treatment ◽

Severe Disease ◽

Neutralizing Antibody ◽

Supplemental Oxygen ◽

European Medicines Agency ◽

Global Public Health ◽

Public Health Burden

The emergence of SARS-CoV-2 and subsequent COVID-19 pandemic has resulted in a significant global public health burden, leading to an urgent need for effective therapeutic strategies. In this article, we review the role of SARS-CoV-2 neutralizing antibodies (nAbs) in the clinical management of COVID-19 and provide an overview of recent randomized controlled trial data evaluating nAbs in the ambulatory, hospitalized and prophylaxis settings. Two nAb cocktails (casirivimab/imdevimab and bamlanivimab/etesevimab) and one nAb monotherapy (bamlanivimab) have been granted Emergency Use Authorization by the US Food and Drug Administration for the treatment of ambulatory patients who have a high risk of progressing to severe disease, and the European Medicines Agency has similarly recommended both cocktails and bamlanivimab monotherapy for use in COVID-19 patients who do not require supplemental oxygen and who are at high risk of progressing to severe COVID-19. Efficacy of nAbs in hospitalized patients with COVID-19 has been varied, potentially highlighting the challenges of antiviral treatment in patients who have already progressed to severe disease. However, early data suggest a promising prophylactic role for nAbs in providing effective COVID-19 protection. We also review the risk of treatment-emergent antiviral resistant “escape” mutants and strategies to minimize their occurrence, discuss the susceptibility of newly emerging SARS-COV-2 variants to nAbs, as well as explore administration challenges and ways to improve patient access.

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Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes

Journal of General Virology ◽

10.1099/vir.0.82456-0 ◽

2007 ◽

Vol 88 (2) ◽

pp. 621-630 ◽

Cited By ~ 151

Author(s):

S. Maan ◽

N. S. Maan ◽

A. R. Samuel ◽

S. Rao ◽

H. Attoui ◽

...

Keyword(s):

Bluetongue Virus ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Full Length ◽

Molecular Diagnostic ◽

Mammalian Host ◽

Reading Frame ◽

Diagnostic Assays ◽

Primary Determinant ◽

Serotype Identification

The outer capsid protein VP2 of Bluetongue virus (BTV) is a target for the protective immune response generated by the mammalian host. VP2 contains the majority of epitopes that are recognized by neutralizing antibodies and is therefore also the primary determinant of BTV serotype. Full-length cDNA copies of genome segment 2 (Seg-2, which encodes VP2) from the reference strains of each of the 24 BTV serotypes were synthesized, cloned and sequenced. This represents the first complete set of full-length BTV VP2 genes (from the 24 serotypes) that has been analysed. Each Seg-2 has a single open reading frame, with short inverted repeats adjacent to conserved terminal hexanucleotide sequences. These data demonstrated overall inter-serotype variations in Seg-2 of 29 % (BTV-8 and BTV-18) to 59 % (BTV-16 and BTV-22), while the deduced amino acid sequence of VP2 varied from 22.4 % (BTV-4 and BTV-20) to 73 % (BTV-6 and BTV-22). Ten distinct Seg-2 lineages (nucleotypes) were detected, with greatest sequence similarities between those serotypes that had previously been reported as serologically ‘related’. Fewer similarities were observed between different serotypes in regions of VP2 that have been reported as antigenically important, suggesting that they may play a role in the neutralizing antibody response. The data presented form an initial basis for BTV serotype identification by sequence analyses and comparison of Seg-2, and for development of molecular diagnostic assays for individual BTV serotypes (by RT-PCR).

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Comparison of Four SARS-CoV-2 Neutralization Assays

Vaccines ◽

10.3390/vaccines9010013 ◽

2020 ◽

Vol 9 (1) ◽

pp. 13

Author(s):

Lydia Riepler ◽

Annika Rössler ◽

Albert Falch ◽

André Volland ◽

Wegene Borena ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Neutralizing Antibodies ◽

The Other ◽

In Vitro Assays ◽

The Novel ◽

Effective Protection ◽

Vaccine Candidates ◽

Vesicular Stomatitis ◽

Novel Coronavirus

Neutralizing antibodies are a major correlate of protection for many viruses including the novel coronavirus SARS-CoV-2. Thus, vaccine candidates should potently induce neutralizing antibodies to render effective protection from infection. A variety of in vitro assays for the detection of SARS-CoV-2 neutralizing antibodies has been described. However, validation of the different assays against each other is important to allow comparison of different studies. Here, we compared four different SARS-CoV-2 neutralization assays using the same set of patient samples. Two assays used replication competent SARS-CoV-2, a focus forming assay and a TCID50-based assay, while the other two assays used replication defective lentiviral or vesicular stomatitis virus (VSV)-based particles pseudotyped with SARS-CoV-2 spike. All assays were robust and produced highly reproducible neutralization titers. Titers of neutralizing antibodies correlated well between the different assays and with the titers of SARS-CoV-2 S-protein binding antibodies detected in an ELISA. Our study showed that commonly used SARS-CoV-2 neutralization assays are robust and that results obtained with different assays are comparable.

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