HIV envelope trimers and gp120 as immunogens to induce broadly neutralizing antibodies in cows.

The study of immunogens capable of eliciting broadly neutralizing antibodies (bnAbs) is crucial for the development of an HIV vaccine. To date, only cows, making use of their ultralong CDRH3 loops, have reliably elicited bnAbs following immunization with HIV Envelope trimers. Antibody responses to the CD4 binding site have been readily elicited by immunization of cows with a stabilized Env trimer of the BG505 strain and, with more difficulty, to the V2-apex region of Env with a cocktail of trimers. Here, we sought to determine whether the BG505 Env trimer could be engineered to generate new bnAb specificities in cows. Since the cow CD4 binding site bnAbs bind to monomeric BG505 gp120, we also sought to determine whether gp120 immunization alone might be sufficient to induce bnAbs. We found that engineering the CD4 binding site by mutation of a key binding residue of BG505 HIV Env resulted in a reduced bnAb response that took more immunizations to develop. Monoclonal antibodies isolated from one animal were directed to the V2-apex, suggesting a re-focusing of the bnAb response. Immunization with monomeric BG505 g120 generated no serum bnAb responses, indicating that the ultralong CDRH3 bnAbs are only elicited in the context of the trimer in the absence of many other less restrictive epitopes presented on monomeric gp120. The results support the notion of a hierarchy of epitopes on HIV Env and suggest that, even with the presence in the cow repertoire of ultralong CDRH3s, bnAb epitopes are relatively disfavored.

Immunization of cows with HIV envelope trimers generates broadly neutralizing antibodies to the V2-apex from the ultralong CDRH3 repertoire.

The generation of broadly neutralizing antibodies (bnAbs) to specific HIV epitopes of the HIV Envelope (Env) is one of the cornerstones of HIV vaccine research. The current animal models we use have been unable to reliable produce a broadly neutralizing antibody response, with the exception of cows. Cows have rapidly and reliably produced a CD4 binding site response by homologous prime and boosting with a native-like Env trimer. In small animal models other engineered immunogens previously have been able to focus antibody responses to the bnAb V2-apex region of Env. Here, we immunized two groups of cows (n=4) with two regiments of V2-apex focusing immunogens to investigate whether antibody responses could be directed to the V2-apex on Env. Group 1 were immunized with chimpanzee simian immunodeficiency virus (SIV)-Env trimer that shares its V2-apex with HIV, followed by immunization with C108, a V2-apex focusing immunogen, and finally boosted with a cross-clade native-like trimer cocktail. Group 2 were immunized with HIV C108 Env trimer followed by the same HIV trimer cocktail as Group 1. Longitudinal serum analysis showed that one cow in each group developed serum neutralizing antibody responses to the V2-apex. Eight and 11 bnAbs were isolated from Group 1 and Group 2 cows respectively. The best bnAbs had both medium breadth and potency. Potent and broad responses developed later than previous CD4bs cow bnAbs and required several different immunogens. All isolated bnAbs were derived from the ultralong CDRH3 repertoire. The finding that cow antibodies can target multiple broadly neutralizing epitopes on the HIV surface reveals important insight into the generation of immunogens and testing in the cow animal model. The exclusive isolation of ultralong CDRH3 bnAbs, despite only comprising a small percent of the cow repertoire, suggests these antibodies outcompete the long and short CDRH3 antibodies during the bnAb response.

The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

Immunization of pigs with replication-incompetent adenovirus-vectored African swine fever virus multi-antigens induced humoral immune responses but no protection following contact challenge.

Introduction: African swine fever virus (ASFV) is a pathogen of great economic importance given that continues to threaten the pork industry worldwide, but there is no safe vaccine or treatment available. Development of a vaccine is feasible as immunization of pigs with some live attenuated ASFV vaccine candidates can confer protection, but safety concerns and virus scalability are challenges that must to be addressed. Identification of protective ASFV antigens is needed to inform the development of efficacious subunit vaccines. Methods: In this study, replication-incompetent adenovirus-vectored multicistronic ASFV antigen expression constructs that covered nearly 100% of the ASFV proteome were generated and validated using ASFV convalescent serum. Swine were immunized with a cocktail of the expression constructs, designated Ad5-ASFV, alone or formulated with either Montanide ISA-201™ (ASFV-ISA-201) or BioMize® adjuvant (ASFV-BioMize). Results: These constructs primed strong B cell responses as judged by anti-pp62-specific IgG responses. Notably, the Ad5-ASFV and the Ad5-ASFV ISA-201, but not the Ad5-ASFV BioMize®, immunogens primed significantly (p < 0.0001) higher anti-pp62-specific IgG responses compared with Ad5-Luciferase formulated with Montanide ISA-201™ adjuvant (Luc-ISA-201). The anti-pp62-specific IgG responses underwent significant (p < 0.0001) recall in all the vaccinees after boosting and the induced antibodies strongly recognized ASFV (Georgia 2007/1)-infected primary swine cells. However, following challenge by contact spreaders, only one pig nearly immunized with the Ad5-ASFV cocktail survived. The survivor had no typical clinical symptoms, but had viral loads and lesions consistent with chronic ASF. Discussion: Besides the limited sample size used, the outcome suggests that in vivo antigen expression, but not the antigen content, might be the limitation of this immunization approach as the replication-incompetent adenovirus does not amplify in vivo to effectively prime and expand protective immunity or directly mimic the gene transcription mechanisms of attenuated ASFV. Addressing the in vivo antigen delivery limitations may yield promising outcomes.

Expression of CD117 (c-Kit) on Circulating B Cells in Pediatric Schistosomiasis.

Few B cells express CD27, the primary marker for memory B cells, in pediatric schistosomiasis, suggesting B cell malfunction. This study further demonstrates unexpected high expression of CD117 on circulating B cells in children highly exposed to Schistosoma mansoni infectious larvae. CD117 is expressed by immature or lymphoma B cells, but not by mature, circulating cells. We therefore sought to define the significance of CD117 on blood B cells. We found that CD117-positive (CD117+) B cells increased with the intensity of schistosome infection. In addition, CD117 expression was reduced on CD23+ B cells previously shown to correlate with resistance to infection. Stimulation with a panel of cytokines demonstrated that CD117 levels were upregulated in response to a combination of interleukin 4 (IL-4) and stem cell factor (SCF), the ligand for CD117, whereas IL-2 led to a reduction. In addition, stimulation with SCF generally reduced B cell activation levels. Upon further investigation, it was established that multiple circulating cells expressed increased levels of CD117, including monocytes, neutrophils, and eosinophils, and expression levels correlated with that of B cells. Finally, we identified a population of large circulating cells with features of reticulocytes. Overall, our results suggest that hyperexposure to intravascular parasitic worms elicits immature cells from the bone marrow. Levels of SCF were shown to reduce as children began to transition through puberty. The study results pose an explanation for the inability of children to develop significant immunity to infection until after puberty.

Absence of lower genital tract lesions among women of reproductive age infected with Schistosoma mansoni: A cross-sectional study using a colposcope in Western Kenya.

BACKGROUND: Female genital schistosomiasis (FGS) constitutes four different lesions known to be caused by Schistosoma haematobium ova deposited in the genital tract. Schistosoma mansoni ova may also be found in the genital tract. However, it is not known if S. mansoni causes lower genital tract lesions characteristic of FGS. METHODOLOGY: This study was conducted in 8 villages along the shores of Lake Victoria, western Kenya. Stool and urine samples, collected from women of reproductive age on three consecutive days, were analysed for S. mansoni and S. haematobium infection. S. mansoni positive and S. haematobium negative willing participants, aged 18-50 years were invited to answer a questionnaire (demographics, symptoms), undergo a gynaecological examination and cytology specimen collection by an FGS expert. PRINCIPAL FINDINGS: Gynaecologic investigations were conducted in 147 S. mansoni-positive women who had a mean infection intensity of 253.3 epg (95% CI: 194.8-311.9 epg). Nearly 90% of them used Lake Victoria as their main water source. None were found to have cervicovaginal grainy sandy patches or rubbery papules. Homogenous yellow patches were found in 12/147 (8.2%) women. Women with homogenous yellow patches were significantly older (47 years) than the rest (34 years, p = 0.001). No association was found between intensity of S. mansoni infection and homogenous yellow patches (p = 0.70) or abnormal blood vessels (p = 0.14). S. mansoni infection intensity was not associated with genital itch, bloody or malodorous vaginal discharge. CONCLUSION: S. mansoni infection was neither associated with lower genital tract lesions nor symptoms typically found in women with FGS.

Adenovirus-Vectored African Swine Fever Virus pp220 Induces Robust Antibody, IFN-γ, and CTL Responses in Pigs.

African Swine Fever Virus (ASFV) poses a serious threat to the pork industry worldwide; however, there is no safe vaccine or treatment available. The development of an efficacious subunit vaccine will require the identification of protective antigens. The ASFV pp220 polyprotein is essential for virus structural integrity. This polyprotein is processed to generate p5, p34, p14, p37, and p150 individual proteins. Immunization of pigs with a cocktail of adenoviruses expressing the proteins induced significant IgG, IFN-γ-secreting cells, and cytotoxic T lymphocyte responses. Four predicted SLA-I binding nonamer peptides, namely p34161-169, p37859-867, p1501363-1371, and p1501463-1471, recalled strong IFN-γ+ PBMC and splenocyte responses. Notably, peptide p34161-169 was recognized by PBMCs isolated from 7/10 pigs and by splenocytes isolated from 8/10 pigs. Peptides p37859-867 and p1501363-1371 stimulated recall IFN-γ+ responses in PBMCs and splenocytes isolated from 8/10 pigs, whereas peptide p1501463-1471 recalled responses in PBMCs and splenocytes isolated from 7/10 to 9/10 pigs, respectively. The results demonstrate that the pp220 polyprotein contains multiple epitopes that induce robust immune responses in pigs. Importantly, these epitopes are 100% conserved among different ASFV genotypes and were predicted to bind multiple SLA-I alleles. The outcomes suggest that pp220 is a promising candidate for inclusion in a prototype subunit vaccine.

Novel Potent IFN-γ-Inducing CD8+ T Cell Epitopes Conserved among Diverse Bovine Viral Diarrhea Virus Strains.

Studies of immune responses elicited by bovine viral diarrhea virus (BVDV) vaccines have primarily focused on the characterization of neutralizing B cell and CD4+ T cell epitopes. Despite the availability of commercial vaccines for decades, BVDV prevalence in cattle has remained largely unaffected. There is limited knowledge regarding the role of BVDV-specific CD8+ T cells in immune protection, and indirect evidence suggests that they play a crucial role during BVDV infection. In this study, the presence of BVDV-specific CD8+ T cells that are highly cross-reactive in cattle was demonstrated. Most importantly, novel potent IFN-γ-inducing CD8+ T cell epitopes were identified from different regions of BVDV polyprotein. Eight CD8+ T cell epitopes were identified from the following structural BVDV Ags: Erns, E1, and E2 glycoproteins. In addition, from nonstructural BVDV Ags Npro, NS2-3, NS4A-B, and NS5A-B, 20 CD8+ T cell epitopes were identified. The majority of these IFN-γ-inducing CD8+ T cell epitopes were found to be highly conserved among more than 200 strains from BVDV-1 and -2 genotypes. These conserved epitopes were also validated as cross-reactive because they induced high recall IFN-γ+CD8+ T cell responses ex vivo in purified bovine CD8+ T cells isolated from BVDV-1- and -2-immunized cattle. Altogether, 28 bovine MHC class I-binding epitopes were identified from key BVDV Ags that can elicit broadly reactive CD8+ T cells against diverse BVDV strains. The data presented in this study will lay the groundwork for the development of a contemporary CD8+ T cell-based BVDV vaccine capable of addressing BVDV heterogeneity more effectively than current vaccines.

Mosaic Bovine Viral Diarrhea Virus Antigens Elicit Cross-Protective Immunity in Calves.

Bovine Viral Diarrhea Virus (BVDV) is an important pathogen that plays a significant role in initiating Bovine Respiratory Disease Complex (BRDC) in cattle. The disease causes multi-billion dollar losses globally due to high calf mortality and increased morbidity leading to heavy use of antibiotics. Current commercial vaccines provide limited cross-protection with several drawbacks such as safety, immunosuppression, potential reversion to virulence, and induction of neonatal pancytopenia. This study evaluates two prototype vaccines containing multiple rationally designed recombinant mosaic BVDV antigens for their potential to confer cross-protection against diverse BVDV strains. Genes encoding three novel mosaic antigens, designated E2123, NS2-31, and NS2-32, were designed in silico and expressed in mammalian cells for the formulation of a prototype protein-based vaccine. The mosaic antigens contain highly conserved protective epitopes from BVDV-1a, -1b, and -2, and included unique neutralizing epitopes from disparate strains to broaden coverage. We tested immunogenicity and protective efficacy of Expi293TM-expressed mosaic antigens (293F-E2123, 293F-NS2-31, and 293F-NS2-32), and baculovirus-expressed E2123 (Bac-E2123) mosaic antigen in calves. The Expi293TM-expressed antigen cocktail induced robust BVDV-specific cross-reactive IFN-γ responses, broadly neutralizing antibodies, and following challenge with a BVDV-1b strain, the calves had significantly (p < 0.05) reduced viremia and clinical BVD disease compared to the calves vaccinated with a commercial killed vaccine. The Bac-E2123 antigen was not as effective as the Expi293TM-expressed antigen cocktail, but it protected calves from BVD disease better than the commercial killed vaccine. The findings support feasibility for development of a broadly protective subunit BVDV vaccine for safe and effective management of BRD.

Progress Toward Development of Effective and Safe African Swine Fever Virus Vaccines.

African swine fever is a major concern due to its negative impact on pork production in affected regions. Due to lack of treatment and a safe vaccine, it has been extremely difficult to control this devastating disease. The mechanisms of virus entry, replication within the host cells, immune evasion mechanisms, correlates of protection, and antigens that are effective at inducing host immune response, are now gradually being identified. This information is required for rational design of novel disease control strategies. Pigs which recover from infection with less virulent ASFV isolates can be protected from challenge with related virulent isolates. This strongly indicates that an effective vaccine against ASFV could be developed. Nonetheless, it is clear that effective immunity depends on both antibody and cellular immune responses. This review paper summarizes the key studies that have evaluated three major approaches for development of African Swine Fever virus vaccines. Recent immunization strategies have involved development and in vivo evaluation of live attenuated virus, and recombinant protein- and DNA-based and virus-vectored subunit vaccine candidates. The limitations of challenge models for evaluating ASFV vaccine candidates are also discussed.

Schistosoma haematobium hotspots in south Nyanza, western Kenya: prevalence, distribution and co-endemicity with Schistosoma mansoni and soil-transmitted helminths.

BACKGROUND: Schistosomiasis studies in western Kenya have mainly focused on the intestinal form, with evidence of urinary schistosomiasis remaining anecdotal. Detailed disease mapping has been carried out predominantly along the shores of Lake Victoria, but there is a paucity of information on intestinal and urinary schistosomiasis in inland sites. METHODS: This cross-sectional survey of 3,487 children aged 7-18 years from 95 schools in south Nyanza, western Kenya determined the prevalence, infection intensity, and geographical distribution of Schistosoma haematobium, evaluating its co-endemicity with Schistosoma mansoni and soil-transmitted helminths (STHs). Helminth eggs were analyzed from single urine (for S. haematobium) and stool (for S. mansoni and STHs) samples by centrifugation and Kato-Katz, respectively. Hematuria was used as a proxy indicator for S. haematobium. Schools and water bodies (ponds, water-points, streams, dams and rivers) were mapped using Geographical Information System and prevalence maps obtained using ArcView GIS Software. RESULTS: S. haematobium infections with an overall prevalence of 9.3% (95% CI = 8.4-10.2%) were mostly prevalent in Rachuonyo, 22.4% (95% CI = 19.2-25.9% and 19.7 eggs/10 ml) and Migori, 10.7% (95% CI = 9.2-12.3% and 29.5 eggs/10 ml) districts, particularly around Kayuka pond and Ongoche river respectively. Overall infections correlated with hematuria (r = 0.9, P < 0.0001) and were more likely in boys (P < 0.0001, OR = 0.624). S. mansoni infections with an overall prevalence of 13% (95% CI =11.9-14.1%) were majorly confined along the shores of Lake Victoria. STH infections were homogenously distributed with A. lumbricoides occurring in 5.4% (95% CI = 4.7-6.3%) and T. trichiura in 2.8% (95% CI = 2.3-3.4%) of the children. Although S. mansoni infections were more co-endemic with S. haematobium, only A. lumbricoides infections were positively associated with S. haematobium (P = 0.0295, OR = 0.4585). Overall prevalence of S. haematobium monoinfection was 7.2% (95% CI = 6.4-8%), S. mansoni monoinfection was 12.3% (95% CI = 10.4-12.5%), and S. haematobium-S. mansoni coinfection was 1.2% (95% CI = 0.9-1.6%). There was no significant difference in infection intensity between mono and coinfections. CONCLUSION: Prevalence distribution maps obtained are important for planning and implementing disease control programs in these areas.