Summary Unlike humans or mice, some species have limited genome encoded combinatorial diversity potential, yet mount a robust antibody response. Cows are unusual in having exceptionally long CDR H3 loops and few V-regions, but the mechanism for creating diversity is not understood. Deep sequencing revealed that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded mini-domains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a β-strand “stalk” that supports a structurally diverse, disulfide-bonded, “knob” domain. Sequence analysis suggests that diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias towards mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of CDR H3s of unprecedented length that fold into a diversity of mini-domains generated through combinations of somatically generated disulfides.

No immunogen to date has reliably elicited broadly neutralizing antibodies (bnAbs) to HIV in humans or animal models. Advances in the design of immunogens (BG505 SOSIP) that antigenically mimic the HIV envelope glycoprotein (Env)1 have improved the elicitation of potent isolate-specific Ab responses in rabbits2 and macaques3, but so far failed to induce bnAbs. One possible contributor to this failure is that the relevant antibody repertoires are poorly suited to target somewhat occluded conserved epitope regions on Env relative to exposed variable epitopes. To test this hypothesis, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach over 70 amino acids in length4–9. Remarkably, BG505 SOSIP immunization resulted in rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and 96% breadth (n = 117) at 381 days. A monoclonal antibody (mAb) isolated from this cow harbored an ultralong HCDR3 of 60 amino acids and neutralized 72% of cross-clade isolates (n = 117) with a potent median IC50 of 0.028 μg/ml. We note that breadth was elicited with a single trimer immunogen and did not require additional envelope diversity. Immunization of cows may provide an avenue to rapidly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.

The antibody repertoire of Bos taurus is characterized by a subset of variable heavy (VH) chain regions with ultralong third complementarity determining regions (CDR3) which, compared to other species, can provide a potent response to challenging antigens like HIV env. These unusual CDR3 can range to over seventy highly diverse amino acids in length and form unique β-ribbon 'stalk' and disulfide bonded 'knob' structures, far from the typical antigen binding site. The genetic components and processes for forming these unusual cattle antibody VH CDR3 are not well understood. Here we analyze sequences of Bos taurus antibody VH domains and find that the subset with ultralong CDR3 exclusively uses a single variable gene, IGHV1-7 (VHBUL) rearranged to the longest diversity gene, IGHD8-2. An eight nucleotide duplication at the 3' end of IGHV1-7 encodes a longer V-region producing an extended F β-strand that contributes to the stalk in a rearranged CDR3. A low amino acid variability was observed in CDR1 and CDR2, suggesting that antigen binding for this subset most likely only depends on the CDR3. Importantly a novel, potentially AID mediated, deletional diversification mechanism of the B. taurus VH ultralong CDR3 knob was discovered, in which interior codons of the IGHD8-2 region are removed while maintaining integral structural components of the knob and descending strand of the stalk in place. These deletions serve to further diversify cysteine positions, and thus disulfide bonded loops. Hence, both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual cattle VH ultralong CDR3 repertoire.Cellular and Molecular Immunology advance online publication, 4 December 2017; doi:10.1038/cmi.2017.117.

Delayed development of virus-specific immune response has been observed in pigs infected with the porcine reproductive and respiratory syndrome virus (PRRSV). Several studies support the hypothesis that the PRRSV is capable of modulating porcine immune system, but the mechanisms involved are yet to be defined. In this study, we evaluated the induction of T regulatory cells by PRRSV-infected dendritic cells (DCs). Our results showed that PRRSV-infected DCs significantly increased Foxp3(+)CD25(+) T cells, an effect that was reversible by IFN-alpha treatment, and this outcome was reproducible using two distinct PRRSV strains. Analysis of the expressed cytokines suggested that the induction of Foxp3(+)CD25(+) T cells is dependent on TGF-beta but not IL-10. In addition, a significant up-regulation of Foxp3 mRNA, but not TBX21 or GATA3, was detected. Importantly, our results showed that the induced Foxp3(+)CD25(+) T cells were able to suppress the proliferation of PHA-stimulated PBMCs. The T cells induced by the PRRSV-infected DCs fit the Foxp3(+)CD25(+) T helper 3 (Th3) regulatory cell phenotype described in the literature. The induction of this cell phenotype depended, at least in part, on PRRSV viability because IFN-alpha treatment or virus inactivation reversed these effects. In conclusion, this data supports the hypothesis that the PRRSV succeeds to establish and replicate in porcine cells early post-infection, in part, by inducing Th3 regulatory cells as a mechanism of modulating the porcine immune system.

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eThe African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (

Acquired T cell immunity is central for protection against infection. However, the immunological consequences of exposing memory T cells to high Ag loads during acute and persistent infection with systemic pathogens are poorly understood. We investigated this by using infection with Anaplasma marginale, a ruminant pathogen that replicates to levels of 109 bacteria per ml of blood during acute infection and maintains mean bacteremia levels of 106 per ml during long-term persistent infection. We established that immunization-induced Ag-specific peripheral blood CD4+ T cell responses were rapidly and permanently lost following infection. To determine whether these T cells were anergic, sequestered in the spleen, or physically deleted from peripheral blood, CD4+ T lymphocytes from the peripheral blood specific for the major surface protein (MSP) 1a T cell epitope were enumerated by DRB3*1101 tetramer staining and FACS analysis throughout the course of immunization and challenge. Immunization induced significant epitope-specific T lymphocyte responses that rapidly declined near peak bacteremia to background levels. Concomitantly, the mean frequency of tetramer+CD4+ cells decreased rapidly from 0.025% before challenge to a preimmunization level of 0.0003% of CD4+ T cells. Low frequencies of tetramer+CD4+ T cells in spleen, liver, and inguinal lymph nodes sampled 9–12 wk postchallenge were consistent with undetectable or unsustainable Ag-specific responses and the lack of T cell sequestration. Thus, infection of cattle with A. marginale leads to the rapid loss of Ag-specific T cells and immunologic memory, which may be a strategy for this pathogen to modulate the immune response and persist.

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.