SARS-CoV-2 is a viral respiratory pathogen responsible for the current global pandemic and the disease that causes COVID-19. All current WHO approved COVID-19 vaccines are administered through the muscular route. We have developed a prototype two-dose vaccine (BReC-CoV-2) by combining the Receptor Binding Domain (RBD) antigen, via conjugation to Diphtheria toxoid (EcoCRM®). The vaccine is adjuvanted with Bacterial Enzymatic Combinatorial Chemistry (BECC), BECC470. Intranasal (IN) administration of BreC-CoV-2 in K18-hACE2 mice induced a strong systemic and localized immune response in the respiratory tissues which provided protection against the Washington strain of SARS-CoV-2. Protection provided after IN administration of BReC-CoV-2 was associated with decreased viral RNA copies in the lung, robust RBD IgA titers in the lung and nasal wash, and induction of broadly neutralizing antibodies in the serum. We also observed that BReC-CoV-2 vaccination administered using an intramuscular (IM) prime and IN boost protected mice from a lethal challenge dose of the Delta variant of SARS-CoV-2. IN administration of BReC-CoV-2 provided better protection than IM only administration to mice against lethal challenge dose of SARS-CoV-2. These data suggest that the IN route of vaccination induces localized immune responses that can better protect against SARS-CoV-2 than the IM route in the upper respiratory tract.

Anaplasma marginale causes bovine anaplasmosis, a debilitating and potentially fatal tick-borne infection of cattle. Because A. marginale is an obligate intracellular organism, its adhesins that mediate entry into host cells are essential for survival. Here, we demonstrate that A. marginale outer membrane protein A (AmOmpA; AM854) contributes to the invasion of mammalian and tick host cells. AmOmpA exhibits predicted structural homology to OmpA of A. phagocytophilum (ApOmpA), an adhesin that uses key lysine and glycine residues to interact with ␣2,3-sialylated and ␣1,3-fucosylated glycan receptors, including 6-sulfo-sialyl Lewis x (6-sulfo-sLe x ). Antisera against AmOmpA or its predicted binding domain inhibits A. marginale infection of host cells. Residues G55 and K58 are contributory, and K59 is essential for recombinant AmOmpA to bind to host cells. Enzymatic removal of ␣2,3-sialic acid and ␣1,3-fucose residues from host cell surfaces makes them less supportive of AmOmpA binding. AmOmpA is both an adhesin and an invasin, as coating inert beads with it confers adhesiveness and invasiveness. Recombinant forms of AmOmpA and ApOmpA competitively antagonize A. marginale infection of host cells, but a monoclonal antibody against 6-sulfo-sLe x fails to inhibit AmOmpA adhesion and A. marginale infection. Thus, the two OmpA proteins bind related but structurally distinct receptors. This study provides a detailed understanding of AmOmpA function, identifies its essential residues that can be targeted by blocking antibody to reduce infection, and determines that it binds to one or more ␣2,3-sialylated and ␣1,3-fucosylated glycan receptors that are unique from those targeted by ApOmpA.

Cyclic-di-GMP (c-di-GMP) contributes to the regulation of processes required by the Lyme disease (LD) spirochetes to complete the tick-mammal enzootic cycle. Our understanding of the effector mechanisms of c-di-GMP in the Borrelia is evolving. While most LD spirochete isolates encode a single PilZ domain containing c-di-GMP receptor designated as PlzA, genome analyses have revealed that a subset encode a second PilZ domain protein (PlzB). The c-di-GMP binding potential of PlzB, and its role in LD spirochete biology, have not been investigated. To determine if PlzB binds c-di-GMP, plzB from B. burgdorferi isolate ZS7 was PCR amplified, cloned, and recombinant protein generated. PlzB bound c-di-GMP but not other nucleotides, indicating a specific binding interaction. To determine if PlzA and PlzB are functionally synonymous, a series of allelic-exchange gene deletion and cis-complemented strains were generated in the B. burgdorferi B31 background. B. burgdorferi B31-ΔplzA was competent to infect Ixodes scapularis larvae but not mice when delivered by either needle or tick feeding. B. burgdorferi B31-ΔplzA also displayed an atypical motility phenotype. Complementation in cis of B. burgdorferi B31-ΔplzA with plzA (B31-plzA KI) restored wild-type (wt) phenotype. However, a strain complemented in cis with plzB (B31-plzB KI) did not. The data presented here are consistent with an earlier study that demonstrated that PlzA plays an essential role in spirochete survival in the mammalian environment. We add to our understanding of the c-di-GMP regulatory network by demonstrating that while PlzB binds c-di-GMP, it is not functionally synonymous with PlzA. The absence of plzB from most strains suggests that it is not required for survival. One possibility is that cells that harbor both PlzA and PlzB might have enhanced biological fitness or increased virulence.

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As Ixodes ticks spread to new regions, the incidence of Lyme disease (LD) in companion animals and humans will increase. Preventive strategies for LD in canines center on vaccination and tick control (acaricides). Both subunit and bacterin based LD veterinary vaccines are available. Outer surface protein C (OspC), a potent immunogen and dominant early antigen, has been demonstrated to elicit protective antibody (Ab) responses. However, a single OspC protein elicits a relatively narrow range of protection. There are conflicting reports as to whether the immunodominant epitopes of OspC reside within variable or conserved domains. A detailed understanding of the antigenic determinants of OspC is essential for understanding immune responses to this essential virulence factor and vaccinogen. Here, we investigate the contribution of the conserved C-terminal C10 motif in OspC triggered Ab responses. Using a panel of diverse recombinant full length OspC proteins and their corresponding C10 deletion variants (OspCΔC10), we demonstrate that the C10 motif does not significantly contribute to immunization or infection induced Ab responses in rabbits, rats, canines, horses and non-human primates. Furthermore, the C10 motif is not required to trigger potent bactericidal Ab responses. This study provides insight into the antigenic structure of OspC. The results enhance our understanding of immune responses that develop during infection or upon vaccination and have implications for interpretation of LD diagnostic assays that employ OspC.