Biphasic MERS-CoV Incidence in Nomadic Dromedaries with Putative Transmission to Humans, Kenya, 2022-2023.

Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in dromedaries in Africa, but camel-to-human transmission is limited. Sustained 12-month sampling of dromedaries in a Kenya abattoir hub showed biphasic MERS-CoV incidence; peak detections occurred in October 2022 and February 2023. Dromedary-exposed abattoir workers (7/48) had serologic signs of previous MERS-CoV exposure.

Detection of African swine fever virus in the tissues of asymptomatic pigs in smallholder farming systems along the Kenya-Uganda border: implications for transmission in endemic areas and ASF surveillance in East Africa.

The persistence of African swine fever virus (ASFV) in endemic areas, with small-scale but regular outbreaks in domestic pigs, is not well understood. ASFV has not been detected using conventional diagnosis in these pigs or adjacent populations of resistant African wild pigs, that could act as potential carriers during the outbreaks. However, such data are crucial for the design of evidence-based control strategies. We conducted cross-sectional (1107 pigs) and longitudinal (100 pigs) monitoring of ASFV prevalence in local pigs in Kenya and Uganda. The horizontal survey revealed no evidence of ASFV in the serum or blood using either conventional or real-time PCR. One pig consistently tested positive using ELISA, but negative using PCR assays on blood. Interestingly, the isotype of the antibodies from this animal were strongly IgA biased relative to control domestic pigs and warthogs, suggesting a role for mucosal immunity. The tissues from this pig were positive by PCR following post-mortem. Internal organ tissues of 44 healthy pigs (28 sentinel pigs and 16 pigs from slaughter slabs) were tested with four different PCR assays; 15.9 % were positive for ASFV suggesting that healthy pigs carrying ASFV exist in the swine population in the study area. P72 and p54 genotyping of ASFV revealed very limited diversity: all were classified in genotype IX at both loci, as were virtually all viruses causing recent ASF outbreaks in the region. Our study suggests that carrier pigs may play a role in ASF disease outbreaks, although the triggers for outbreaks remain unclear and require further investigation. This study significantly increases scientific knowledge of the epidemiology of ASF in the field in Africa, which will contribute to the design of effective surveillance and control strategies.

Biomarkers of mortality in adults and adolescents with advanced HIV in sub-Saharan Africa.

One-third of people with HIV in sub-Saharan Africa start antiretroviral therapy (ART) with advanced disease. We investigated associations between immune biomarkers and mortality in participants with advanced HIV randomised to cotrimoxazole or enhanced antimicrobial prophylaxis in the Reduction of Early Mortality in HIV-Infected Adults and Children Starting Antiretroviral Therapy (REALITY) trial (ISRCTN43622374). Biomarkers were assayed using ELISA and Luminex. Associations between baseline values and all-cause 24-week mortality were analysed using Cox models, and for cause-specific mortality used Fine & Gray models, including prophylaxis randomisation, viral load, CD4, WHO stage, age, BMI, and site as covariates; and weighted according to inverse probability of selection into the substudy. Higher baseline CRP, IFN-γ, IL-6 and IP-10 were associated with higher all-cause mortality; and higher IL-23, IL-2 and RANTES with lower all-cause mortality. Associations varied by cause of death: tuberculosis-associated mortality was most strongly associated with higher CRP and sST2, and cryptococcosis-associated mortality with higher IL-4 and lower IL-8. Changes in I-FABP (p = 0.002), faecal alpha-1 antitrypsin (p = 0.01) and faecal myeloperoxidase (p = 0.005) between baseline and 4 weeks post-ART were greater in those receiving enhanced versus cotrimoxazole prophylaxis. Our findings highlight how the immune milieu shapes outcomes following ART initiation, and how adjunctive antimicrobials can modulate the gut environment in advanced HIV.

The African Swine Fever Isolate ASFV-Kenya-IX-1033 Is Highly Virulent and Stable after Propagation in the Wild Boar Cell Line WSL.

We describe the characterization of an African swine fever genotype IX virus (ASFV-Kenya-IX-1033), which was isolated from a domestic pig in western Kenya during a reported outbreak. This includes the efficiency of virus replication and in vivo virulence, together with genome stability and virulence, following passage in blood macrophages and in a wild boar lung cell line (WSL). The ASFV-Kenya-IX-1033 stock retained its ability to replicate in primary macrophages and retained virulence in vivo, following more than 20 passages in a WSL. At the whole genome level, a few single-nucleotide differences were observed between the macrophage and WSL-propagated viruses. Thus, we propose that the WSL is suitable for the production of live-attenuated ASFV vaccine candidates based on the modification of this wild-type isolate. The genome sequences for ASFV-Kenya-IX-1033 propagated in macrophages and in WSL cells were submitted to GenBank, and a challenge model based on the isolate was developed. This will aid the development of vaccines against the genotype IX ASFV circulating in eastern and central Africa.

Allele frequency-based and polymorphism-versus-divergence indices of balancing selection in a new filtered set of polymorphic genes in Plasmodium falciparum.

Signatures of balancing selection operating on specific gene loci in endemic pathogens can identify candidate targets of naturally acquired immunity. In malaria parasites, several leading vaccine candidates convincingly show such signatures when subjected to several tests of neutrality, but the discovery of new targets affected by selection to a similar extent has been slow. A small minority of all genes are under such selection, as indicated by a recent study of 26 Plasmodium falciparum merozoite-stage genes that were not previously prioritized as vaccine candidates, of which only one (locus PF10_0348) showed a strong signature. Therefore, to focus discovery efforts on genes that are polymorphic, we scanned all available shotgun genome sequence data from laboratory lines of P. falciparum and chose six loci with more than five single nucleotide polymorphisms per kilobase (including PF10_0348) for in-depth frequency-based analyses in a Kenyan population (allele sample sizes >50 for each locus) and comparison of Hudson-Kreitman-Aguade (HKA) ratios of population diversity (π) to interspecific divergence (K) from the chimpanzee parasite Plasmodium reichenowi. Three of these (the msp3/6-like genes PF10_0348 and PF10_0355 and the surf(4.1) gene PFD1160w) showed exceptionally high positive values of Tajima's D and Fu and Li's F indices and have the highest HKA ratios, indicating that they are under balancing selection and should be prioritized for studies of their protein products as candidate targets of immunity. Combined with earlier results, there is now strong evidence that high HKA ratio (as well as the frequency-independent ratio of Watterson's /K) is predictive of high values of Tajima's D. Thus, the former offers value for use in genome-wide screening when numbers of genome sequences within a species are low or in combination with Tajima's D as a 2D test on large population genomic samples.

Bovine Pestivirus Heterogeneity and Its Potential Impact on Vaccination and Diagnosis.

Bovine Pestiviruses A and B, formerly known as bovine viral diarrhoea viruses (BVDV)-1 and 2, respectively, are important pathogens of cattle worldwide, responsible for significant economic losses. Bovine viral diarrhoea control programmes are in effect in several high-income countries but less so in low- and middle-income countries where bovine pestiviruses are not considered in disease control programmes. However, bovine pestiviruses are genetically and antigenically diverse, which affects the efficiency of the control programmes. The emergence of atypical ruminant pestiviruses (Pestivirus H or BVDV-3) from various parts of the world and the detection of Pestivirus D (border disease virus) in cattle highlights the challenge that pestiviruses continue to pose to control measures including the development of vaccines with improved cross-protective potential and enhanced diagnostics. This review examines the effect of bovine pestivirus diversity and emergence of atypical pestiviruses in disease control by vaccination and diagnosis.

Plasma calprotectin as a biomarker of mortality at antiretroviral treatment initiation in advanced HIV - pilot study.

Background: In advanced HIV, significant mortality occurs soon after starting antiretroviral treatment (ART) in low- and middle-incomes countries. Calprotectin is a biomarker of innate response to infection and inflammatory conditions. We examined the association between plasma calprotectin at initiation of ART and mortality among individuals with advanced HIV. Methods: We conducted a pilot case-cohort study among HIV infected adults and children over 5 years old with CD4 + <100/mm 3 at ART initiation at two Kenyan sites. Participants received three factorial randomised interventions in addition to ART within the REALITY trial ( ISRCTN43622374). Calprotectin was measured by ELISA in archived plasma of those who died within 24 weeks (cases) and randomly selected participants who survived for 48 weeks (non-cases) for whom samples were available. Factors associated with baseline plasma calprotectin were investigated using linear regression. To test association with mortality, Cox proportional hazards models with inverse sampling probability weights and adjusted for age, sex, site, BMI, viral load, randomised treatments, and clustered by CD4 count were fitted. Results: Baseline median (IQR) plasma calprotectin was 6.82 (2.65-12.5) µg/ml in cases (n=39) and 5.01 (1.92-11.5) µg/ml in non-cases (n=58). Baseline calprotectin was associated with age, neutrophil count and the presence of cough, but not other measured indicators of infection. In adjusted multivariable models, baseline calprotectin was associated with subsequent mortality: HR 1.64 (95% CI 1.11 - 2.42) and HR 2.77 (95% CI 1.58 - 4.88) for deaths during the first twenty-four and four weeks respectively. Calprotectin levels fell between baseline and 4 weeks among both cases and non-cases irrespective of randomised interventions. Conclusion: Among individuals with advanced HIV starting ART in Kenya, plasma calprotectin may have potential as a biomarker of early mortality. Validation in larger studies, comparison with other biomarkers and investigation of the sources of infection and inflammation are warranted.

Characterization of the fine specificity of bovine CD8 T-cell responses to defined antigens from the protozoan parasite Theileria parva.

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes.

Comparative analysis of adaptive immune responses following experimental infections of cattle with bovine viral diarrhoea virus-1 and an Asiatic atypical ruminant pestivirus.

Atypical ruminant pestiviruses are closely related to the two bovine viral diarrhoea virus (BVDV) species, BVDV-1 and BVDV-2. While there is evidence of cross-protective immune responses between BVDV-1 and BVDV-2, despite antigenic differences, there is little information on the antigenic cross-reactivity with atypical ruminant pestiviruses. The aim of this study was therefore to assess the specificity of antibody and T cell responses induced by experimental infection of calves with BVDV-1 strain Ho916, Th/04_KhonKaen (TKK), an Asiatic atypical ruminant pestivirus, or co-infection with both viruses. Homologous virus neutralization was observed in sera from both single virus infected and co-infected groups, while cross-neutralization was only observed in the TKK infected group. T cell IFN-γ responses to both viruses were observed in the TKK infected animals, whereas Ho916 infected calves responded better to homologous virus. Specifically, IFN-γ responses to viral non-structural protein, NS3, were observed in all infected groups while responses to viral glycoprotein, E2, were virus-specific. Broader antigen-specific cytokine responses were observed with similar trends between inoculation groups and virus species. The limited T cell and antibody immune reactivity of Ho916 inoculated animals to TKK suggests that animals vaccinated with current BVDV-1-based vaccines may not be protected against atypical ruminant pestiviruses.

Design and evaluation of the immunogenicity and efficacy of a biomimetic particulate formulation of viral antigens.

Subunit viral vaccines are typically not as efficient as live attenuated or inactivated vaccines at inducing protective immune responses. This paper describes an alternative 'biomimetic' technology; whereby viral antigens were formulated around a polymeric shell in a rationally arranged fashion with a surface glycoprotein coated on to the surface and non-structural antigen and adjuvant encapsulated. We evaluated this model using BVDV E2 and NS3 proteins formulated in poly-(D, L-lactic-co-glycolic acid) (PLGA) nanoparticles adjuvanted with polyinosinic:polycytidylic acid (poly(I:C) as an adjuvant (Vaccine-NP). This Vaccine-NP was compared to ovalbumin and poly(I:C) formulated in a similar manner (Control-NP) and a commercial adjuvanted inactivated BVDV vaccine (IAV), all inoculated subcutaneously and boosted prior to BVDV-1 challenge. Significant virus-neutralizing activity, and E2 and NS3 specific antibodies were observed in both Vaccine-NP and IAV groups following the booster immunisation. IFN-γ responses were observed in ex vivo PBMC stimulated with E2 and NS3 proteins in both vaccinated groups. We observed that the protection afforded by the particulate vaccine was comparable to the licenced IAV formulation. In conclusion, the biomimetic particulates showed a promising immunogenicity and efficacy profile that may be improved by virtue of being a customisable mode of delivery.

Assessment of the enhancement of PLGA nanoparticle uptake by dendritic cells through the addition of natural receptor ligands and monoclonal antibody.

Targeting of specific receptors on antigen-presenting cells is an appealing prospect in the production of novel nanoparticulate vaccines. In particular, the targeting of vaccines to dendritic cell (DC) subsets has been shown in models to significantly improve the induction of immune responses. This paper describes the evaluation of natural ligands, mannan and chitosan, and monoclonal antibodies as targeting motifs to enhance uptake of PLGA nanoparticle carriers by bovine DCs. To assess enhancement of uptake after the addition of natural ligands a bovine monocyte derived DC (MoDC) model was used. For the assessment of monoclonal antibody targeting, the model was expanded to include afferent lymph DCs (ALDCs) in a competitive uptake assay. Mannan, proved unsuccessful at enhancing uptake or targeting by MoDCs. Chitosan coated particle uptake could be impeded by the addition of mannan suggesting uptake may be mediated through sugar receptors. Inclusion of monoclonal antibodies specific for the DEC-205 (CD205) receptor increased the number of receptor expressing DCs associated with particles as well as the number of particles taken up by individual cells. These results support the further evaluation of active targeting of nanovaccines to DCs to enhance their immunogenicity in cattle and other large mammalian species including humans.

Kinetics of single and dual infection of calves with an Asian atypical bovine pestivirus and a highly virulent strain of bovine viral diarrhoea virus 1.

Atypical bovine pestiviruses related to bovine viral diarrhoea virus (BVDV) have recently been detected in cattle from South America, Asia and Europe. The purpose of this study was to compare the clinical and virological aspects of dual infection with BVDV-1 (Horton 916) and an Asian atypical bovine pestivirus (Th/04_KhonKaen) in naïve calves, in comparison to single infections. Milder clinical signs were observed in the animals infected with single Th/04_KhonKaen strain. Leukocytopenia and lymphocytopenia were observed in all infected groups at a similar level which correlated with the onset of viraemia. Co-infection with both viruses led to prolonged fever in comparison to single strain inoculated groups and simultaneous replication of concurrent viruses in blood and in the upper respiratory tract. Following the infections all the calves seroconverted against homologous strains. Atypical pestiviruses pose a serious threat to livestock health and BVDV eradication, since they may have the potential to be widely spread in cattle populations without being detected and differentiated from other BVDV infections.