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Vibrio cholerae O1 is one of two serogroups responsible for epidemic cholera, a severe watery diarrhea that occurs after the bacterium colonizes the human small intestine and secretes a potent ADP-ribosylating toxin. Immunity to cholera is associated with intestinal anti-lipopolysaccharide (LPS) antibodies, which are known to inhibit V. cholerae motility and promote bacterial cell-cell crosslinking and aggregation. Here we report that V. cholerae O1 classical and El Tor biotypes produce an extracellular matrix (ECM) when forcibly immobilized and agglutinated by ZAC-3 IgG, an intestinally-derived monoclonal antibody (MAb) against the core/lipid A region of LPS. ECM secretion, as demonstrated by crystal violet staining and scanning electron microscopy, occurred within 30 minutes of antibody exposure and peaked by 3 hours. Non-motile mutants of V. cholerae did not secrete ECM following ZAC-3 IgG exposure, even though they were susceptible to agglutination. The ECM was enriched in O-specific polysaccharide (OSP) but not Vibrio polysaccharide (VPS). Finally, we demonstrate that ECM production by V. cholerae in response to ZAC-3 IgG was associated with bacterial resistant to a secondary complement-mediated attack. In summary, we propose that V. cholerae O1, upon encountering anti-LPS antibodies in the intestinal lumen, secretes an ECM (or O-antigen capsule) possibly as a strategy to shield itself from additional host immune factors and to exit an otherwise inhospitable host environment.

•ZAC-3 is a monoclonal antibody against a conserved Vibrio cholerae epitope.•ZAC-3 is an inhibitor of V. cholerae flagellum-based motility.•ZAC-3 IgG and Fab fragments reduce V. cholerae intestinal colonization in a mouse model.•ZAC-3 reveals potential of antibodies to confer immunity across V. cholerae biotypes. Vibrio cholerae is the causative agent of cholera, an acute diarrheal disease that remains endemic in many parts of the world. The mechanisms underlying immunity to cholera remain poorly defined, though it is increasingly clear that protection is associated with antibodies against lipopolysaccharide (LPS). Here we report that ZAC-3, a monoclonal antibody against the core/lipid A region of V. cholerae LPS is a potent inhibitor of V. cholerae flagellum-based motility in viscous and liquid environments. ZAC-3 arrested motility of the classical Ogawa strain O395, as well as the El Tor Inaba strain C6706. In addition, we demonstrate, in the neonatal mouse model, that ZAC-3 IgG and Fab fragments significantly reduced the ability of both V. cholerae strains O395 and C6706 to colonize the intestinal epithelium, revealing the potential of antibodies against the core/lipid A to contribute to immunity across biotypes, possibly through a mechanism involving motility arrest.

ABSTRACT Following an episode of cholera, a rapidly dehydrating, watery diarrhea caused by the Gram-negative bacterium, Vibrio cholerae O1, humans mount a robust anti-lipopolysaccharide (LPS) antibody response that is associated with immunity to subsequent re-infection. In neonatal mouse and rabbit models of cholera, passively administered anti-LPS polyclonal and monoclonal (MAb) antibodies reduce V. cholerae colonization of the intestinal epithelia by inhibiting bacterial motility and promoting vibrio agglutination. Here we demonstrate that human anti-LPS IgG MAbs also arrest V. cholerae motility and induce bacterial paralysis. A subset of those MAbs also triggered V. cholerae to secrete an extracellular matrix (ECM). To identify changes in gene expression that accompany antibody exposure and that may account for motility arrest and ECM production, we subjected V. cholerae O1 El Tor to RNA-seq analysis after treatment with ZAC-3 IgG, a high affinity MAb directed against the core/lipid A region of LPS. We identified > 160 genes whose expression was altered following ZAC-3 IgG treatment, although canonical outer membrane stress regulons were not among them. ompS (VCA1028), a porin associated with virulence and indirectly regulated by ToxT, and norR (VCA0182), a σ54-dependent transcription factor involved in late stages of infection, were two upregulated genes worth noting. Vibrio cholerae alters gene expression upon encountering mucosal antibodies.

Background/Objectives: We aimed to determine safe and immunogenic RSVpreF vaccine dose levels for further clinical development in 2–<18 year olds. Methods: The phase 1, age-descending, open-label Picasso trial evaluated different RSVpreF dose levels in respiratory syncytial virus (RSV)-seropositive 2–<5 year olds and 5–<18 year olds who were either healthy or had chronic medical conditions with increased RSV illness risk. Participants received a single dose of RSVpreF (60 µg or 120 µg dose level). The primary objective was to describe safety and tolerability at each dose level and age group, including frequencies of reactogenicity and adverse events (AEs). The secondary objective was to describe RSV neutralizing antibody responses at each dose level and age group 1 month after vaccination. Results: Overall, 127 participants received RSVpreF 60 µg (2–<5 year olds, n = 20; 5–<18 year olds, n = 35) or 120 µg (n = 24 and n = 48, respectively); 54% were male and 69% were White. Local reactions and systemic events were reported in 17–20% and 33–45% of 2–<5 year olds, respectively, and 49–56% and 52–60% of 5–<18 year olds; most were mild or moderate in severity. AEs were reported in 13–15% of 2–<5 year olds and 8–14% of 5–<18 year olds. No AEs leading to withdrawal or vaccine-related serious AEs were reported. RSV-A and RSV-B neutralizing titer geometric mean fold rises from before to 1 month after vaccination with RSVpreF 60 and 120 µg, which were 17.7–20.6 and 42.8–39.8, respectively, in 2–<5 year olds, and 19.0–23.5 and 20.3–20.3, respectively, in 5–<18 year olds. Conclusions: RSVpreF was safe, well tolerated, and elicited immune responses in RSV-seropositive 2–<18-year-old participants, supporting further clinical development in this pediatric population, including those with chronic conditions.

Lyme disease is a growing public health concern that is geographically focused in regions where ticks that carry the causative bacteria, Borrelia burgdorferi sensu lato (s.l.), are endemic. Outer surface protein A (OspA) is expressed by B. burgdorferi s.l. spirochetes during the tick phase and OspA antibodies introduced during tick feeding can block transmission and prevent B. burgdorferi infection. Candidate Lyme disease vaccine VLA15 is comprised of the C-terminal domains of the six B. burgdorferi s.l. OspA serotypes (ST) prevalent in North America and Europe. We report herein that non-human primates immunized with VLA15 were protected against challenge with Ixodes scapularis ticks bearing B. burgdorferi sensu stricto (s.s.) (OspA ST1). Levels of residual B. burgdorferi s.s. tick colonization were reduced in ticks that fed on VLA15-immunized primates compared to those immunized with full length-OspA ST1 (FL-OspA) at a point when OspA-binding IgG levels were similar. Furthermore, monoclonal antibodies targeting the C-terminal half of OspA, elicited by FL-OspA immunization in primates, were more effective at complement-mediated bactericidal killing in vitro and clearance of spirochetes in ticks versus those directed against other parts of the protein. •VLA15 immunization protected NHPs against challenge with Ixodes ticks bearing B. burgdorferi ss (OspA ST1).•Post-challenge tick colonization levels were reduced in VLA15-immunized NHPs compared to levels in FL-OspA-immunized NHPs, despite similar anti-OspA IgG levels between the two vaccine groups.•Monoclonal antibodies targeting the C-terminal and central domains of OspA ST1 bound cell-associated OspA and manifested greater functional activity in vitro, and in vivo in mice challenged with Ixodes ticks bearing B. burgdorferi ss.

The outer membrane (OM) of Gram-negative enteric pathogens like Vibrio cholerae is a barrier against host defense factors, as well as a sensor of physical and chemical stimuli that the bacteria encounter in the gastrointestinal tract. The OM is also the primary target of the mucosal immune response, which consists of secretory antibodies primarily directed against lipopolysaccharide (LPS). ZAC-3 is a monoclonal antibody (MAb) that targets the conserved core/lipid A region of LPS of the pandemic V. cholerae O1 serotype. In a neonatal mouse model, passively administered ZAC-3 IgG has been shown to reduce the ability of V. cholerae to colonize the intestinal epithelium. In cholera epidemics, the spread of disease can easily outpace vaccine control measures. The advent of technologies enabling the expression of recombinant proteins, including antibodies, in the milk of transgenic animals enables the development of a self-administered and cost-effective MAb-based prophylactic to reduce the incidence of V. cholerae infection. In Chapter 3, I demonstrate that transgenic mice can express ZAC-3 as a human IgA1 in their milk. This ZAC-3 hIgA retained binding to V. cholerae LPS and the ability to inhibit flagellum-mediated motility. Additionally, neonates fed from dams secreting ZAC-3 hIgA1 show significant reduction in colonization of both classical and El Tor strains of V. cholerae O1, with most mice having no recoverable colony forming units in their intestines at 24 h post-gavage. Having further supported ZAC-3’s efficacy in reduction of colonization we wanted to further study the response of V. cholerae to ZAC-3 binding. In vitro, ZAC-3 IgG is a potent inhibitor of V. cholerae flagellum-based motility and bacterial agglutination, which have been attributed as major drivers of immunity in vivo. However, scanning electron microscopy previously revealed that ZAC-3 IgG also induces changes to the OM that include blebbing and the formation of web-like extensions between cells. We reasoned, therefore, that ZAC-3 IgG may impart stress on the OM and, in turn, impact bacterial pathogenesis. In Chapter 4, I demonstrate that V. cholerae O1 induces a novel extracellular matrix (ECM) in response to ZAC-3 binding, which is dependent on agglutination and motility arrest. This ECM is enriched in LPS and independent from previously characterized Vibrio polysaccharide (VPS) containing biofilm regulation. I demonstrate that the ECM functions like a capsule, as it decreased susceptibility of V. cholerae to lytic assaults on the membrane. I conclude that the ECM response to ZAC-3 IgG is possibly a defensive strategy employed to resist secondary attack by antimicrobial peptides or other host factors in the gut. To further examine the effect of ZAC-3 IgG on integrity of the cell envelope and impacts on bacterial motility, in Chapter 5 I tested the hypothesis that ZAC-3 binding results in a depletion of membrane potential. One mechanism by which membrane potential could be impacted is via activation of inner membrane (IM) embedded mechanosensitive channels of small conductance (MscS). MscS proteins are a member of a family of tension activated channels that enable osmolyte and solute release under mechanical stress. I demonstrated that membrane energetics were not significantly changed by ZAC-3 antibody treatment at concentrations associated with complete motility arrest. Furthermore, ZAC-3 treatment had no effect on bacterial doubling time, a further indicator that membrane potential was unaltered by ZAC-3 exposure. Finally, I demonstrated that deletion mutations in two of the five MscS family genes in V. cholerae, VcmscS (VC0480) and VCA0181, have no alteration in response to ZAC-3. However, both genes encode functional tension-activated channels and VcMscS significantly contributes to survival of hypoosmotic shock. Together these data demonstrate that V. cholerae undergoes antibody induced motility arrest and OM blebbing without alteration of cell division or membrane potential. To investigate the possibility that motility arrest and ECM production in response to ZAC-3 IgG are regulated at the transcriptional level, in Chapter 6, we subjected V. cholerae O1 to RNA-sequencing analysis. One hour of ZAC-3 treatment resulted in differential regulation of 164 genes. However, I show here that major virulence factors, toxin-coregulated pilus (TCP) and cholera toxin (CTx), which are essential for colonization and disease respectively, were unperturbed in gene regulation and protein production by ZAC-3 treatment. This suggests that while ZAC-3 impacts motility, a major virulence factor impacting localization and dissemination in the lumen of the intestine, and gene expression, with implications for modulating a myriad of cellular processes, it does not impact major virulence factor production. Together these data indicate that while colonization is significantly inhibited in the presence of ZAC-3, likely due to motility arrest and possibly ECM induction, cells maintain the ability to divide and produce major virulence factors. This suggests that V. cholerae O1 adapts to ZAC-3 binding in ways that may significantly impact pathogenesis, while not inhibiting critical cellular processes if bacteria are able to escape antibody binding or are shed back into the environment. In addition, ZAC-3 induced ECM production and alteration of OM mechano-physiology have significant implications on bacterial survival in various environmental conditions, including transmission from the host into freshwater environments. This work underscores the importance of the integration of immune status on the study of bacterial pathogenesis.