A nucleic acid-based orthopoxvirus vaccine targeting the vaccinia virus L1, A27, B5 and A33 proteins protects rabbits against lethal rabbitpox virus aerosol challenge

In the age of COVID, nucleic acid vaccines have garnered much attention, at least in part, because of the simplicity of construction, production, and flexibility to adjust and adapt to an evolving outbreak. Orthopoxviruses remain a threat on multiple fronts, especially as emerging zoonosis. In response, we developed a DNA vaccine, termed 4pox, that protected nonhuman primates against monkeypox virus (MPXV) induced severe disease. Here, we examined the protective efficacy of the 4pox DNA vaccine delivered by intramuscular (i.m.) electroporation (EP) in rabbits challenged with aerosolized rabbitpox virus (RPXV), a model that recapitulates the respiratory route of exposure and low dose associated with natural smallpox exposure in humans.
We found that 4pox vaccinated rabbits developed immunogen-specific antibodies, including neutralizing antibodies and did not develop any clinical disease, indicating protection against aerosolized RPXV. In contrast, unvaccinated animals developed significant signs of disease, including lesions, and were euthanized. These findings demonstrate that an unformulated, non-adjuvanted DNA vaccine delivered (i.m.) can protect against an aerosol exposure.
Importance The eradication of smallpox and subsequent cessation of vaccination has left a majority of the population susceptible to variola virus or other emerging poxvirus. This is exemplified by human monkeypox, as evidenced by the increase in reported endemic and imported cases over the past decades.
Therefore, a malleable vaccine technology that can be mass-produced, and doesn’t require complex conditions for distribution and storage is sought. Herein, we show that a DNA vaccine, in the absence of a specialized formulation or adjuvant, can protect against a lethal aerosol insult of rabbitpox virus.

Asymptomatic Orthopoxvirus Circulation in Humans in the Wake of a Monkeypox Outbreak among Chimpanzees in Cameroon

Monkeypox virus is a zoonotic Orthopoxvirus (OPXV) that causes smallpox-like illness in humans. In Cameroon, human monkeypox cases were confirmed in 2018, and outbreaks in captive chimpanzees occurred in 2014 and 2016. We investigated the OPXV serological status among staff at a primate sanctuary (where the 2016 chimpanzee outbreak occurred) and residents from nearby villages, and describe contact with possible monkeypox reservoirs. We focused specifically on Gambian rats (Cricetomys spp.) because it is a recognized possible reservoir and because contact with this species was common enough to render sufficient statistical power.
We collected one 5-mL whole blood specimen from each participant to perform a generic anti-OPXV ELISA test for IgG and IgM antibodies and administered a questionnaire about prior symptoms of monkeypox-like illness and contact with possible reservoirs. Our results showed evidence of OPXV exposures (IgG positive, 6.3%; IgM positive, 1.6%) among some of those too young to have received smallpox vaccination (born after 1980, n = 63). No participants reported prior symptoms consistent with monkeypox.
After adjusting for the education level, participants who frequently visited the forest were more likely to have recently eaten Gambian rats (OR: 3.36, 95% CI: 1.91-5.92, P < 0.001) and primate sanctuary staff were less likely to have touched or sold Gambian rats (OR: 0.23, 95% CI: 0.19-0.28, P < 0.001). The asymptomatic or undetected circulation of OPXVs in humans in Cameroon is likely, and contact with monkeypox reservoirs is common, raising the need for continued surveillance for human and animal diseases.

Co-administration of tecovirimat and ACAM2000 in non-human primates: Effect of tecovirimat treatment on ACAM2000 immunogenicity and efficacy versus lethal monkeypox virus challenge.

Naturally occurring smallpox has been eradicated but research stocks of variola virus (VARV), the causative agent of smallpox, still exist in secure laboratories. Clandestine stores of the virus or resurrection of VARV via synthetic biology are possible and have led to concerns that VARV could be used as a biological weapon. The US government has prepared for such an event by stockpiling smallpox vaccines and TPOXX, SIGA Technologies’ smallpox antiviral drug. While vaccination is effective as a pre-exposure prophylaxis, protection is limited when administered following exposure. Safety concerns preclude general use of the vaccine unless there is a smallpox outbreak.
TPOXX is approved by the FDA for use after confirmed diagnosis of smallpox disease. Tecovirimat, the active pharmaceutical ingredient in TPOXX, targets a highly conserved orthopoxviral protein, inhibiting long-range dissemination of virus. Although indications for use of the vaccine and TPOXX do not overlap, concomitant use is possible, especially if the TPOXX indication is expanded to include post-exposure prophylaxis.
It is therefore important to understand how vaccine and TPOXX may interact. In studies presented here, monkeys were vaccinated with the ACAM2000TM live attenuated smallpox vaccine and concomitantly treated with tecovirimat or placebo. Immune responses to the vaccine and protective efficacy versus a lethal monkeypox virus (MPXV) challenge were evaluated. In two studies, primary and anamnestic humoral immune responses were similar regardless of tecovirimat treatment while the third study showed reduction in vaccine elicited humoral immunity.
Following lethal MPXV challenge, all (12 of 12) vaccinated/placebo treated animals survived, and 12 of 13 vaccinated/tecovirimat treated animals survived. Clinical signs of disease were elevated in tecovirimat treated animals compared to placebo treated animals. This suggests that TPOXX may affect the immunogenicity of ACAM2000 if administered concomitantly. These studies may inform on how vaccine and TPOXX are used during a smallpox outbreak.

Use of live Variola virus to determine whether CAST/EiJ mice are a suitable surrogate animal model for human smallpox

Numerous animal models of systemic orthopoxvirus disease have been developed to evaluate therapeutics against variola virus (VARV), the causative agent of smallpox. These animal models do not resemble the disease presentation in human smallpox and most used surrogate Orthopoxviruses. A rodent model using VARV has a multitude of advantages, and previous investigations identified the CAST/EiJ mouse as highly susceptible to monkeypox virus infection, making it of interest to determine if these rodents are also susceptible to VARV infection.

Recombivirus Monkey Anti-monkeypox IgG ELISA kit, 96 tests, Quantitative

AE-311150-1 Alpha Diagnostics 1 kit

Recombivirus? Monkey Anti-Zika Virus (ZIKV) Envelop protein IgG ELISA kit, 96 tests, Quantitative

RV-403110-1 Alpha Diagnostics 1 Kit

Recombivirus? Monkey Anti-Zika Virus (ZIKV) PrM protein IgG ELISA kit, 96 tests, Quantitative

RV-403210-1 Alpha Diagnostics 1 Kit

Recombivirus? Monkey Anti-Zika Virus (ZIKV) NS1 protein IgG ELISA kit, 96 tests, Quantitative

RV-403310-1 Alpha Diagnostics 1 Kit

Recombivirus? Monkey Anti-Zika Virus (ZIKV) Capsid protein IgG ELISA kit, 96 tests, Quantitative

RV-403410-1 Alpha Diagnostics 1 Kit

Ibrutinib drug-Human IgG Conjugate

IBT17-IgG Alpha Diagnostics 100 ug

Rabbit Anti-Monkey IgG

FNSA-0099 FN Test 500 uL

Goat anti Monkey IgG

40R-IG002 Fitzgerald 1 mg

Goat anti Monkey IgG

41C-CB1605 Fitzgerald 1 mg

Rabbit Polyclonal antibody Anti-CRBN

Anti-CRBN ImmunoStep 50 µg

Recombivirus Rabbit Anti-rabbitpox (RPXV090) IgG ELISA kit, 96 tests

AE-311120-1 Alpha Diagnostics 1 kit

Recombivirus Horse Anti-horsepox IgG ELISA kit, 96 tests, Quantitative

AE-311140-1 Alpha Diagnostics 1 kit

Recombivirus? Monkey Anti-Zika Virus (ZIKV) Envelop domain III IgG ELISA kit, 96 tests, Quantitative

RV-403160-1 Alpha Diagnostics 1 Kit

RecombiVirus Monkey EDIM/rotavirus VP6 antibody IgG ELISA Kit, 96 tests, Quantitative

AE-300440-1 Alpha Diagnostics 1 kit

Monoclonal Mouse Anti-Monkey IgG

C010205-10mg Unibiotest 10mg

Monoclonal Mouse Anti-Monkey IgG

C010205-1mg Unibiotest 1mg

Mouse Anti-Monkey IgG antibody

STJ99551-100l St John's Laboratory 100 µl

Mouse Anti-Monkey IgG antibody

STJ99551-1mL St John's Laboratory 1 mL

Rabbit anti Rhesus Monkey IgG

FNSA-0092 FN Test 500 uL

Goat anti Monkey IgG (HRP)

43R-IG020HRP Fitzgerald 1 mg

Goat anti Monkey IgG (biotin)

43R-IG021bt Fitzgerald 1 mg

Goat anti Monkey IgG (FITC)

43R-IG022ft Fitzgerald 1 mg

Goat anti Monkey IgG (rhodamine)

43R-IG054RD Fitzgerald 1 mg

Recombivirus Monkey Corona virus (Bcov/BCV) nucleocapsid protein IgG ELISA kit, 96 tests

RV-500530-1 Alpha Diagnostics 1 kit

Rabbit Anti-Monkey IgG(h+l)

C020217-100mg Unibiotest 100mg

Rabbit Anti-Monkey IgG(h+l)

C020217-1mg Unibiotest 1mg

HRP*Monoclonal Mouse Anti-Monkey IgG

C030204-10ml Unibiotest 10ml

HRP*Monoclonal Mouse Anti-Monkey IgG

C030204-1ml Unibiotest 1ml

FITC*Monoclonal Mouse Anti-Monkey IgG

C030604-10ml Unibiotest 10ml

FITC*Monoclonal Mouse Anti-Monkey IgG

C030604-1ml Unibiotest 1ml

BIOTIN*Monoclonal Mouse Anti-Monkey IgG

C030804-10ml Unibiotest 10ml

BIOTIN*Monoclonal Mouse Anti-Monkey IgG

C030804-1ml Unibiotest 1ml

Rabbit Anti Monkey Igg Polyclonal Antibody

DPBT-67277RM Creative Diagnostics 5 ml

Mouse Anti-Monkey IgG antibody (AU)

STJ99381-100l St John's Laboratory 100 µl

Mouse Anti-Monkey IgG antibody (AU)

STJ99381-1mL St John's Laboratory 1 mL

Mouse Anti-Monkey IgG antibody (Biotin)

STJ99431-100l St John's Laboratory 100 µl

Mouse Anti-Monkey IgG antibody (Biotin)

STJ99431-1mL St John's Laboratory 1 mL

Mouse Anti-Monkey IgG antibody (FITC)

STJ99481-100l St John's Laboratory 100 µl

Mouse Anti-Monkey IgG antibody (FITC)

STJ99481-1mL St John's Laboratory 1 mL

Mouse Anti-Monkey IgG antibody (HRP)

STJ99531-100l St John's Laboratory 100 µl

Mouse Anti-Monkey IgG antibody (HRP)

STJ99531-1mL St John's Laboratory 1 mL

Rabbit Anti Monkey IgG (H+L)

FNSA-0098 FN Test 500 uL

Monkey Anti-Paragonimus IgG ELISA kit

E09A2101-192T BlueGene 192 tests

Monkey Anti-Paragonimus IgG ELISA kit

E09A2101-48 BlueGene 1 plate of 48 wells

Monkey Anti-Paragonimus IgG ELISA kit

E09A2101-96 BlueGene 1 plate of 96 wells

Rabbit anti Monkey IgG (H + L)

40C-CB1600 Fitzgerald 1 mg

Goat anti Monkey IgG (H + L)

40C-CB1601 Fitzgerald 1 mg

Goat anti Monkey IgG (Alk Phos)

43C-CB1611 Fitzgerald 1 mg

Mouse Anti-Monkey IgG Antibody [5D12A12]

PM-6341 ProSci 0.05 mg

Monkey anti cardiolipin antibody IgG,ACA IgG ELISA kit

E09A2051-192T BlueGene 192 tests

Monkey anti cardiolipin antibody IgG,ACA IgG ELISA kit

E09A2051-48 BlueGene 1 plate of 48 wells

Monkey anti cardiolipin antibody IgG,ACA IgG ELISA kit

E09A2051-96 BlueGene 1 plate of 96 wells

Monkey Anti tissue transglutaminase IgG,IgG tTG ELISA kit

E09A2131-192T BlueGene 192 tests

Monkey Anti tissue transglutaminase IgG,IgG tTG ELISA kit

E09A2131-48 BlueGene 1 plate of 48 wells

Monkey Anti tissue transglutaminase IgG,IgG tTG ELISA kit

E09A2131-96 BlueGene 1 plate of 96 wells

Recombivirus? Monkey Anti-Zika Virus (ZIKV) Env+NS1+Prm+Capsid antibody Combo IgG ELISA kit, 96 tests, Quantitative

RV-403460-C Alpha Diagnostics 1 Kit

RecombiVirus Mouse Parvovirus (MPV) IgG ELISA Kit, 96 tests

AE-300500-1 Alpha Diagnostics 1 kit

RecombiVirus Mouse Anti-Seneca Valley Virus (SeV) IgG ELISA kit, Quantitative, 96 tests

RV-404220-1 Alpha Diagnostics 1 kit

Rabbit Anti-Monkey IgG (H&L) Antibody

20-abx134868 Abbexa
  • 100 ul
  • 500 ul

Mouse Anti-Monkey IgG (H&L) Antibody

20-abx134869 Abbexa
  • 100 ul
  • 500 ul

Mouse Anti-Monkey IgG (H&L) Antibody

20-abx134870 Abbexa
  • 100 ul
  • 500 ul

Goat anti-Monkey IgG (H/L) Antibody

abx401231-1mg Abbexa 1 mg

Rabbit Anti-Monkey IgG (H+L) antibody

STJ99569-100l St John's Laboratory 100 µl

Rabbit Anti-Monkey IgG (H+L) antibody

STJ99569-1mL St John's Laboratory 1 mL

Goat Anti-Human IgG (Monkey Adsorbed), unlabeled

10419-UL Alpha Diagnostics 250 ug

Monkey Anti myocardial antibody IgG ELISA kit

E09A0560-192T BlueGene 192 tests

Monkey Anti myocardial antibody IgG ELISA kit

E09A0560-48 BlueGene 1 plate of 48 wells

Monkey Anti myocardial antibody IgG ELISA kit

E09A0560-96 BlueGene 1 plate of 96 wells

Monkey Anti nucleosome antibody IgG ELISA kit

E09A0829-192T BlueGene 192 tests

Monkey Anti nucleosome antibody IgG ELISA kit

E09A0829-48 BlueGene 1 plate of 48 wells

Monkey Anti nucleosome antibody IgG ELISA kit

E09A0829-96 BlueGene 1 plate of 96 wells

Monkey anti gliadin antibody (IgG) ELISA kit

E09A2073-192T BlueGene 192 tests

Monkey anti gliadin antibody (IgG) ELISA kit

E09A2073-48 BlueGene 1 plate of 48 wells

Monkey anti gliadin antibody (IgG) ELISA kit

E09A2073-96 BlueGene 1 plate of 96 wells

Goat anti Monkey IgG (H + L) (FITC)

43C-CB1602 Fitzgerald 1 mg

Goat anti Monkey IgG (H + L) (HRP)

43C-CB1603 Fitzgerald 1 mg

Mouse Anti-Monkey IgG Antibody [5D12A12] (HRP)

1605 ProSci 0.05 mg

Mouse Anti-Monkey IgG Antibody [5D12A12] (biotin)

1606 ProSci 0.05 mg

Polyclonal Goat anti-GST α-form

GST-ANTI-1 Detroit R&D 50 uL

Polyclonal Goat anti-GST μ-form

GST-ANTI-2 Detroit R&D 50 uL

Polyclonal Goat anti-GST p-form

GST-ANTI-3 Detroit R&D 50 uL

Recombivirus? Camel Anti-Peste des petits ruminants NP (PPR-NP) IgG positive control serum

RV-400820-06P Alpha Diagnostics 1 ml

Recombivirus? Human Anti-Zika Virus (ZIKV) Envelop protein IgG ELISA kit, 96 tests, Quantitative

RV-403100-1 Alpha Diagnostics 1 Kit

Recombivirus? Mouse Anti-Zika Virus (ZIKV) Envelop protein IgG ELISA kit, 96 tests, Quantitative

RV-403120-1 Alpha Diagnostics 1 Kit

Recombivirus? Rabbit Anti-Zika Virus (ZIKV) Envelop protein IgG ELISA kit, 96 tests, Quantitative

RV-403130-1 Alpha Diagnostics 1 Kit

Recombivirus? Human Anti-Zika Virus (ZIKV) PrM protein IgG ELISA kit, 96 tests, Quantitative

RV-403200-1 Alpha Diagnostics 1 Kit

Recombivirus? Mouse Anti-Zika Virus (ZIKV) PrM protein IgG ELISA kit, 96 tests, Quantitative

RV-403220-1 Alpha Diagnostics 1 Kit

Recombivirus? Rabbit Anti-Zika Virus (ZIKV) prM protein IgG ELISA kit, 96 tests, Quantitative

RV-403230-1 Alpha Diagnostics 1 Kit

Recombivirus? Human Anti-Zika Virus (ZIKV) NS1 protein IgG ELISA kit, 96 tests, Quantitative

RV-403300-1 Alpha Diagnostics 1 Kit

Recombivirus? Mouse Anti-Zika Virus (ZIKV) NS1 protein IgG ELISA kit, 96 tests, Quantitative

RV-403320-1 Alpha Diagnostics 1 Kit

Recombivirus? Rabbit Anti-Zika Virus (ZIKV) NS1 protein IgG ELISA kit, 96 tests, Quantitative

RV-403330-1 Alpha Diagnostics 1 Kit

Recombivirus? Human Anti-Zika Virus (ZIKV) Capsid protein IgG ELISA kit, 96 tests, Quantitative

RV-403400-1 Alpha Diagnostics 1 Kit

Recombivirus? Mouse Anti-Zika Virus (ZIKV) Capsid protein IgG ELISA kit, 96 tests, Quantitative

RV-403420-1 Alpha Diagnostics 1 Kit

Recombivirus? Rabbit Anti-Zika Virus (ZIKV) Capsid protein IgG ELISA kit, 96 tests, Quantitative

RV-403430-1 Alpha Diagnostics 1 Kit

Recombivirus Mouse Anti-Cytomegalovirus (MCMV/MuHV-1) gB IgG ELISA Kit, 96 tests, Quantitative

AE-301000-1 Alpha Diagnostics 1 Kit

RecombiVirus Rat Minute Virus (MVM) IgG ELISA Kit, 96 tests

AE-300910-1 Alpha Diagnostics 1 kit

RecombiVirus Rat sialodacryoadenitis Virus (SDAV) IgG ELISA Kit, 96 tests

AE-310310-1 Alpha Diagnostics 1 kit

RecombiVirus Rat Pneumonia Virus (PVM) IgG ELISA Kit, 96 tests

AE-310410-1 Alpha Diagnostics 1 kit

Recombivirus Mouse Hantavirus Nucleoprotein (HVNP) IgG ELISA Kit, 96 tests

AE-310600-1 Alpha Diagnostics 1 kit

Recombivirus Rat Hantavirus Nucleoprotein (HVNP) IgG ELISA Kit, 96 tests

AE-310610-1 Alpha Diagnostics 1 kit
In this study, we inoculated CAST/EiJ mice with a range of VARV doses (102-106 plaque forming units). Some animals had detectable viable VARV from the oropharynx between days 3 and 12 post inoculation. Despite evidence of disease, the CAST/EiJ mouse does not provide a model for clinical smallpox due to mild signs of morbidity and limited skin lesions.
However, in contrast to previous rodent models using VARV challenge (i.e. prairie dogs and SCID mice), a robust immune response was observed in the CAST/EiJ mice (measured by Immunoglobulin G enzyme-linked immunosorbent assay). This is an advantage of this model for the study of VARV and presents a unique potential for the study of the immunomodulatory pathways following VARV infection.

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