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Serological assessment of antibody levels is a crucial measure of immunity in vaccinated animals. Establishing the level of antibodies considered protective is essential for vaccine standardization and evaluation of efficacy. The virus neutralization test (VNT), recognized as the gold standard for detecting virus-specific antibodies able to neutralize virus. This study evaluated the effect of viral dose on the detection of humoral immune responses in rabbits vaccinated with a cowpox virus-based vaccine. Blood serum samples were collected on days 14, 21, and 28 post-vaccination. VNT was conducted using viral doses of 100, 50, 25, and 10 TCID . Additionally, the infectious dose 50 (ID ) of the challenge virus was determined based on the induction of skin necrosis in 50% of infected animals. This dose (316 ID per 0.1 mL) was then used to challenge vaccinated rabbits in order to determine the protective antibody titer threshold. Lower viral doses (25 and 10 TCID ) demonstrated higher sensitivity, with neutralizing antibody titers detected at 1:16 and above, significantly exceeding those obtained using 50 and 100 TCID . Based on these findings, 25 TCID was selected as the optimal dose for future VNT. Following cowpox virus challenge, rabbits with neutralizing titers ≥1:16 were protected from skin necrosis, while non-immunized animals developed characteristic lesions. These results suggest that a low-dose (25 TCID ) VNT improves the sensitivity and that a titer of 1:16 can be considered a protective threshold. This approach provides a reliable laboratory model for assessing the immunogenicity and efficacy of cowpox virus vaccines. The results obtained in this study allow for an objective assessment of the immunity elicited from a cowpox vaccine using a laboratory model.

Costimulation is required for optimal T cell activation, yet it is unclear whether poxviruses dedicatedly subvert costimulation during infection. Here, we report that the secreted M2 protein encoded by cowpox virus (CPXV) specifically interacts with human and murine B7.1 (CD80) and B7.2 (CD86). We also show that M2 competes with CD28 and CTLA4 for binding to cell surface B7 ligands, with stronger efficacy against CD28. Functionally, recombinant M2 and culture supernatants from wild-type (WT) but not M2-deficient (ΔM2) CPXV-infected cells can potently suppress B7 ligand-mediated T cell proliferation and interleukin-2 (IL-2) production. Furthermore, we observed increased antiviral CD4 and CD8 T cell responses in C57BL/6 mice challenged by ΔM2 CPXV compared with WT virus. These differences in immune responses to ΔM2 and WT CPXV were not observed in CD28-deficient mice. Taken together, our findings define a mechanism of viral sabotage of T cell activation that highlights the role of CD28 costimulation in host defense against poxvirus infections.

Mass vaccination has played a critical role in the global eradication of smallpox. Various vaccinia virus (VACV) strains, whose origin has not been clearly documented in most cases, have been used as live vaccines in different countries. These VACV strains differed in pathogenicity towards various laboratory animals and in reactogenicity exhibited upon vaccination of humans. In this work, we studied the development of humoral and cellular immune responses in BALB/c mice inoculated intranasally (i.n.) or intradermally (i.d.) with the VACV LIVP strain at a dose of 105 PFU/mouse, which was used in Russia as the first generation smallpox vaccine. Active synthesis of VACV-specific IgM in the mice occurred on day 7 after inoculation, reached a maximum on day 14, and decreased by day 29. Synthesis of virus-specific IgG was detected only from day 14, and the level increased significantly by day 29 after infection of the mice. Immunization (i.n.) resulted in significantly higher production of VACV-specific antibodies compared to that upon i.d. inoculation of LIVP. There were no significant differences in the levels of the T cell response in mice after i.n. or i.d. VACV administration at any time point. The maximum level of VACV-specific T-cells was detected on day 14. By day 29 of the experiment, the level of VACV-specific T-lymphocytes in the spleen of mice significantly decreased for both immunization procedures. On day 30 after immunization with LIVP, mice were infected with the cowpox virus at a dose of 46 LD50. The i.n. immunized mice were resistant to this infection, while 33% of i.d. immunized mice died. Our findings indicate that the level of the humoral immune response to vaccination may play a decisive role in protection of animals from orthopoxvirus reinfection.

Cowpox virus infections in captive cheetahs (Acinonyx jubatus) with high morbidity and mortality have already been reported in the UK and Russia in the 1970s. However, most of the reported cases have been singular events. Here, we report a total of five cowpox virus outbreaks in cheetahs in the same safari park in Denmark between 2010 and 2014. Nine cheetahs showed varying severity of clinical disease; two of them died (22%). All episodes occurred between August and October of the respective year. No other carnivores kept at the same institution nor the keepers taking care of the animals were clinically affected. The clinical picture of cowpox was confirmed by extensive laboratory investigations including histopathological and molecular analyses as well as cell culture isolation of a cowpox virus. High anti-orthopoxvirus antibody titers were detected in all 9 diseased cheetahs compared to seven contact cheetahs without clinical signs and 13 cheetahs not in direct contact. Additionally, whole genome sequencing from one sample of each cluster with subsequent phylogenetic analysis showed that the viruses from different outbreaks have individual sequences but clearly form a clade distinct from other cowpox viruses. However, the intra-clade distances are still larger than those usually observed within clades of one event. These findings indicate multiple and separate introductions of cowpox virus, probably from wild rodent populations, where the virus keeps circulating naturally and is only sporadically introduced into the cheetahs. Sero-positivity of voles (Arvicola amphibious) caught in zoo grounds strengthens this hypothesis. As a consequence, recommendations are given for medical and physical management of diseased cheetahs, for hygienic measures as well as for pre-shipment isolation before cheetah export from zoo grounds.

Four cowpox virus (CPXV) outbreaks occurred in unrelated alpaca herds in Eastern Germany during 2012–2017. All incidents were initially noticed due to severe, generalized, and finally lethal CPXV infections, which were confirmed by testing of tissue and serum samples. As CPXV-infection has been described in South American camelids (SACs) only three times, all four herds were investigated to gain a deeper understanding of CPXV epidemiology in alpacas. The different herds were investigated twice, and various samples (serum, swab samples, and crusts of suspicious pox lesions, feces) were taken to identify additionally infected animals. Serum was used to detect CPXV-specific antibodies by performing an indirect immunofluorescence assay (iIFA); swab samples, crusts, and feces were used for detection of CPXV-specific DNA in a real-time PCR. In total, 28 out of 107 animals could be identified as affected by CPXV, by iIFA and/or PCR. Herd seroprevalence ranged from 16.1% to 81.2%. To investigate the potential source of infection, wild small mammals were trapped around all alpaca herds. In two herds, CPXV-specific antibodies were found in the local rodent population. In the third herd, CPXV could be isolated from a common vole (Microtus arvalis) found drowned in a water bucket used to water the alpacas. Full genome sequencing and comparison with the genome of a CPXV from an alpaca from the same herd reveal 99.997% identity, providing further evidence that the common vole is a reservoir host and infection source of CPXV. Only in the remaining fourth herd, none of the trapped rodents were found to be CPXV-infected. Rodents, as ubiquitous reservoir hosts, in combination with increasingly popular alpacas, as susceptible species, suggest an enhanced risk of future zoonotic infections.

Although viral MHC class I inhibition is considered a classic immune-evasion strategy, its in vivo role is largely unclear. Mutant cowpox virus lacking its MHC class I inhibitors is markedly attenuated during acute infection because of CD8 ⁺ T-cell–dependent control, but it was not known how CD8 ⁺ T-cell responses are affected. Interestingly, we found no major effect of MHC class I down-regulation on priming of functional cowpox virus-specific CD8 ⁺ T cells. Instead, we demonstrate that, during acute infection in vivo, MHC class I down-regulation prevents primed virus-specific CD8 ⁺ T cells from recognizing infected cells and exerting effector responses to control the infection.

One of the hallmarks of viral immune evasion is the capacity to disrupt major histocompatibility complex class I (MHCI) antigen presentation to evade T-cell detection. Cowpox virus encoded protein CPXV203 blocks MHCI surface expression by exploiting the KDEL-receptor recycling pathway, and here we show that CPXV203 directly binds a wide array of fully assembled MHCI proteins, both classical and non-classical. Further, the stability of CPXV203/MHCI complexes is highly pH dependent, with dramatically increased affinities at the lower pH of the Golgi relative to the endoplasmic reticulum (ER). Crystallographic studies reveal that CPXV203 adopts a beta-sandwich fold similar to poxvirus chemokine binding proteins, and binds the same highly conserved MHCI determinants located under the peptide-binding platform that tapasin, CD8, and natural killer (NK)-receptors engage. Mutagenesis of the CPXV203/MHCI interface identified the importance of two CPXV203 His residues that confer low pH stabilization of the complex and are critical to ER retrieval of MHCI. These studies clarify mechanistically how CPXV203 coordinates with other cowpox proteins to thwart antigen presentation.

Most hosts, including humans, are simultaneously or sequentially infected with several parasites. A key question is whether patterns of coinfection arise because infection by one parasite species affects susceptibility to others or because of inherent differences between hosts. We used time-series data from individual hosts in natural populations to analyze patterns of infection risk for a microparasite community, detecting large positive and negative effects of other infections. Patterns remain once variations in host susceptibility and exposure are accounted for. Indeed, effects are typically of greater magnitude, and explain more variation in infection risk, than the effects associated with host and environmental factors more commonly considered in disease studies. We highlight the danger of mistaken inference when considering parasite species in isolation rather than parasite communities.

For almost 150 years after Edward Jenner had published the “Inquiry” in 1798, it was generally assumed that the cowpox virus was the vaccine against smallpox. It was not until 1939 when it was shown that vaccinia, the smallpox vaccine virus, was serologically related but different from the cowpox virus. In the absence of a known natural host, vaccinia has been considered to be a laboratory virus that may have originated from mutational or recombinational events involving cowpox virus, variola viruses or some unknown ancestral Orthopoxvirus. A favorite candidate for a vaccinia ancestor has been the horsepox virus. Edward Jenner himself suspected that cowpox derived from horsepox and he also believed that “matter” obtained from either disease could be used as preventative of smallpox. During the 19th century, inoculation with cowpox (vaccination) was used in Europe alongside with inoculation with horsepox (equination) to prevent smallpox. Vaccine-manufacturing practices during the 19th century may have resulted in the use of virus mixtures, leading to different genetic modifications that resulted in present-day vaccinia strains. Horsepox, a disease previously reported only in Europe, has been disappearing on that continent since the beginning of the 20th century and now seems to have become extinct, although the virus perhaps remains circulating in an unknown reservoir. Genomic sequencing of a horsepox virus isolated in Mongolia in 1976 indicated that, while closely related to vaccinia, this horsepox virus contained additional, potentially ancestral sequences absent in vaccinia. Recent genetic analyses of extant vaccinia viruses have revealed that some strains contain ancestral horsepox virus genes or are phylogenetically related to horsepox virus. We have recently reported that a commercially produced smallpox vaccine, manufactured in the United States in 1902, is genetically highly similar to horsepox virus, providing a missing link in this 200-year-old mystery.

The transmission dynamics of the cowpox virus infection have been quantified in two mixed populations of bank voles (Clethrionomys glareolus) and wood mice (Apodemus sylvaticus), through analyses of detailed time-series of the numbers of susceptible, infectious and newly infected individuals. The cowpox virus is a zoonosis which circulates in these rodent hosts and has been shown to have an adverse effect on reproductive output. The transmission dynamics within species is best described as frequency dependent rather than density dependent, contrary to the 'mass action' assumption of most previous studies, both theoretical and empirical. Estimation of a transmission coefficient for each species in each population also allows annual and seasonal variations in transmission dynamics to be investigated through an analysis of regression residuals. Transmission between host species is found to be negligible despite their close co-habitation. The consequences of this for the combining ability of hosts as zoonotic reservoirs, and for apparent competition between hosts, are discussed.