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Due to its high transmissibility, Klebsiella pneumoniae (Kpn) is one of the leading causes of nosocomial infections. Here, we studied the biological cost of colistin resistance, an antibiotic of last resort, of this opportunistic pathogen using a murine model of gut colonization and transmission. Colistin resistance in Kpn is commonly the result of inactivation of the small regulatory protein MgrB. Without a functional MgrB, the two-component system PhoPQ is constitutively active, leading to increased lipid A modifications and subsequent colistin resistance. Using an engineered MgrB mutant, we observed that MgrB-dependent colistin resistance is not associated with a fitness defect during in vitro growth conditions. However, colistin-resistant Kpn colonizes the murine gut poorly, which may be due to the decreased production of capsular polysaccharide by the mutant. The colistin-resistant mutant of Kpn had increased survival outside the host when compared to the parental colistin-sensitive strain. We attribute this enhanced survivability to dysregulation of the PhoPQ two-component system and accumulation of the master stress regulator RpoS. The enhanced survival of the colistin resistant strain may be a key factor in the observed rapid host-to-host transmission in our model. Together, our data demonstrate that colistin-resistant Kpn experiences a biological cost in gastrointestinal colonization. However, this cost is mitigated by enhanced survival outside the host, increasing the risk of transmission. Additionally, it underscores the importance of considering the entire life cycle of a pathogen to truly determine the biological cost associated with antibiotic resistance.ImportanceThe biological cost associated with colistin resistance in Klebsiella pneumoniae (Kpn) was examined using a murine model of Kpn gut colonization and fecal-oral transmission. A common mutation resulting in colistin resistance in Kpn is a loss-of-function mutation of the small regulatory protein MgrB that regulates the two-component system PhoPQ. Even though colistin resistance in Kpn comes with a fitness defect in gut colonization, it increases bacterial survival outside the host enabling it to more effectively transmit to a new host. The enhanced survival is dependent upon the accumulation of RpoS and dysregulation of the PhoPQ. Hence, our study expands our understanding of the underlying molecular mechanism contributing to the transmission of colistin-resistant Kpn.

The development of an effective AIDS vaccine remains a challenge. Nucleoside-modified mRNAs formulated in lipid nanoparticles (mRNA-LNP) have proved to be a potent mode of immunization against infectious diseases in preclinical studies, and are being tested for SARS-CoV-2 in humans. A critical question is how mRNA-LNP vaccine immunogenicity compares to that of traditional adjuvanted protein vaccines in primates. Here, we show that mRNA-LNP immunization compared to protein immunization elicits either the same or superior magnitude and breadth of HIV-1 Env-specific polyfunctional antibodies. Immunization with mRNA-LNP encoding Zika premembrane and envelope or HIV-1 Env gp160 induces durable neutralizing antibodies for at least 41 weeks. Doses of mRNA-LNP as low as 5 μg are immunogenic in macaques. Thus, mRNA-LNP can be used to rapidly generate single or multi-component vaccines, such as sequential vaccines needed to protect against HIV-1 infection. Such vaccines would be as or more immunogenic than adjuvanted recombinant protein vaccines in primates.

SummarySARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infectionin vitro, while five non-neutralizing NTD antibodies mediated FcγR-independentin vitroinfection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, whilein vitroantibody-enhanced infection does not necessarily herald enhanced infectionin vivo, increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.

Development of an effective AIDS vaccine remains a challenge. Nucleoside-modified mRNAs formulated in lipid nanoparticles (mRNA-LNP) have proved to be a potent mode of immunization against infectious diseases in preclinical studies, and are being tested for SARS-CoV-2 in humans. A critical question is how mRNA-LNP vaccine immunogenicity compares to that of traditional adjuvanted protein vaccines in primates. Here, we found that mRNA-LNP immunization compared to protein immunization elicited either the same or superior magnitude and breadth of HIV-1 Env-specific polyfunctional antibodies. Immunization with mRNA-LNP encoding Zika premembrane and envelope (prM-E) or HIV-1 Env gp160 induced durable neutralizing antibodies for at least 41 weeks. Doses of mRNA-LNP as low as 5 μg were immunogenic in macaques. Thus, mRNA-LNP can be used to rapidly generate single or multi-component vaccines, such as sequential vaccines needed to protect against HIV-1 infection. Such vaccines would be as or more immunogenic than adjuvanted recombinant protein vaccines in primates.

Efforts to eliminate MTCT of HIV with antiretroviral therapy (ART) have met little success, with >180,000 infant infections each year worldwide. It is therefore likely that additional immunologic strategies that can synergize with ART will be required to eliminate MTCT of HIV. To this end, understanding the role of maternal HIV Env-specific IgG antibodies in the setting of MTCT is crucial. In this study, we found that maternal-plasma broadly neutralizing antibody (bNAb) responses can select for T/F viruses that initiate infection in infants. We propose that clinical trials testing the efficacy of single bNAb specificities should not include HIV-infected pregnant women, as a single bNAb might select for neutralization-resistant infant-T/F viruses., Each year, >180,000 infants become infected via mother-to-child transmission (MTCT) of HIV despite the availability of effective maternal antiretroviral treatments, underlining the need for a maternal HIV vaccine. We characterized 224 maternal HIV envelope (Env)-specific IgG monoclonal antibodies (MAbs) from seven nontransmitting and transmitting HIV-infected U.S. and Malawian mothers and examined their neutralization activities against nontransmitted autologous circulating viruses and infant-transmitted founder (infant-T/F) viruses. Only a small subset of maternal viruses, 3 of 72 (4%), were weakly neutralized by maternal linear V3 epitope-specific IgG MAbs, whereas 6 out of 6 (100%) infant-T/F viruses were neutralization resistant to these V3-specific IgG MAbs. We also show that maternal-plasma broadly neutralizing antibody (bNAb) responses targeting the V3 glycan supersite in a transmitting woman may have selected for an N332 V3 glycan neutralization-resistant infant-T/F virus. These data have important implications for bNAb-eliciting vaccines and passively administered bNAbs in the setting of MTCT.

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques., Graphical abstract, Convalescent human-derived SARS-CoV-2 RBD and NTD antibodies mediated neutralization as well as infection enhancement in vitro, yet infusion of these antibodies in mice or cynomolgus macaques resulted in suppression of virus replication.

A primary goal of HIV-1 vaccine development is the consistent elicitation of protective, neutralizing antibodies. While highly similar neutralizing antibodies (nAbs) have been isolated from multiple HIV-infected individuals, it is unclear whether vaccination can consistently elicit highly similar nAbs in genetically diverse primates. Here, we show in three outbred rhesus macaques that immunization with Env elicits a genotypically and phenotypically conserved nAb response. From these vaccinated macaques, we isolated four antibody lineages that had commonalities in immunoglobulin variable, diversity, and joining gene segment usage. Atomic-level structures of the antigen binding fragments of the two most similar antibodies showed nearly identical paratopes. The Env binding modes of each of the four vaccine-induced nAbs were distinct from previously known monoclonal HIV-1 neutralizing antibodies, but were nearly identical to each other. The similarities of these antibodies show that the immune system in outbred primates can respond to HIV-1 Env vaccination with a similar structural and genotypic solution for recognizing a particular neutralizing epitope. These results support rational vaccine design for HIV-1 that aims to reproducibly elicit, in genetically diverse primates, nAbs with specific paratope structures capable of binding conserved epitopes., Author summary The primate immune system generates a diverse repertoire of pathogen-binding proteins called antibodies. Primate antibody repertoires must be diverse to respond to infection by many different pathogens. In this study, we characterized the structure and genetic features of HIV-1 inhibitory antibodies from vaccinated monkeys as a model for human vaccination. We found that individual monkeys responded to HIV vaccination by generating highly similar HIV-1 neutralizing antibodies. The antibodies had nearly identical structures and genetic makeups. Thus, the immune systems of different monkeys generate a common solution for inhibiting viral protein function and infectivity. Success of vaccines in multiple recipients may be augmented by the ability of immune systems to reproducibly devise a common antibody solution to attack vulnerable sites on pathogens.

Each year, over 180,000 infants become infected via mother-to-child-transmission (MTCT) of HIV despite the wide availability of highly effective maternal antiretroviral treatments, underlining the need for a maternal HIV vaccine to end the pediatric HIV epidemic. Here, we characterized 225 maternal HIV Envelope (Env)-specific IgG monoclonal antibodies (mAbs) from seven U.S. and Malawian HIV-infected non-transmitting and transmitting mothers and examined their neutralization activity against autologous circulating non-transmitted viruses and infant transmitted/founder (T/F) viruses. Maternal linear V3 epitope-specific IgG mAbs neutralized maternal circulating non-transmitted viruses yet their paired infant T/F viruses were neutralization resistant to these V3-specific IgG mAbs. We also report that maternal plasma broadly neutralizing antibody (bNAb) responses targeting the N332 V3 glycan supersite in a transmitting woman selected for an N332 V3 glycan neutralization-resistant infant T/F virus, demonstrating that vertical transmission can occur in the presence of maternal plasma bNAb responses. These data have important implications for vaccination strategies aimed at eliciting bNAbs as well as ongoing clinical trials testing the viral-suppressive or prophylactic-potential of passively administered bNAbs in the setting of MTCT.

The ALVAC prime/ALVAC + AIDSVAX B/E boost RV144 vaccine trial induced an estimated 31% efficacy in a low-risk cohort where HIV‑1 exposures were likely at mucosal surfaces. An immune correlates study demonstrated that antibodies targeting the V2 region and in a secondary analysis antibody-dependent cellular cytotoxicity (ADCC), in the presence of low envelope-specific (Env-specific) IgA, correlated with decreased risk of infection. Thus, understanding the B cell repertoires induced by this vaccine in systemic and mucosal compartments are key to understanding the potential protective mechanisms of this vaccine regimen. We immunized rhesus macaques with the ALVAC/AIDSVAX B/E gp120 vaccine regimen given in RV144, and then gave a boost 6 months later, after which the animals were necropsied. We isolated systemic and intestinal vaccine Env-specific memory B cells. Whereas Env-specific B cell clonal lineages were shared between spleen, draining inguinal, anterior pelvic, posterior pelvic, and periaortic lymph nodes, members of Env‑specific B cell clonal lineages were absent in the terminal ileum. Env‑specific antibodies were detectable in rectal fluids, suggesting that IgG antibodies present at mucosal sites were likely systemically produced and transported to intestinal mucosal sites.