Search

The repeat emergence of SARS-CoV-2 variants of concern (VoC) with decreased susceptibility to vaccine-elicited antibodies highlights the need to develop next-generation vaccine candidates that confer broad protection. Here we describe the antibody response induced by the SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine candidate adjuvanted with the Army Liposomal Formulation including QS21 (ALFQ) in non-human primates. By isolating and characterizing several monoclonal antibodies directed against the Spike Receptor Binding Domain (RBD), N-Terminal Domain (NTD), or the S2 Domain, we define the molecular recognition of vaccine-elicited cross-reactive monoclonal antibodies (mAbs) elicited by SpFN. We identify six neutralizing antibodies with broad sarbecovirus cross-reactivity that recapitulate serum polyclonal antibody responses. In particular, RBD mAb WRAIR-5001 binds to the conserved cryptic region with high affinity to sarbecovirus clades 1 and 2, including Omicron variants, while mAb WRAIR-5021 offers complete protection from B.1.617.2 (Delta) in a murine challenge study. Our data further highlight the ability of SpFN vaccination to stimulate cross-reactive B cells targeting conserved regions of the Spike with activity against SARS CoV-1 and SARS-CoV-2 variants., (© 2024. The Author(s).)

The need for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) next-generation vaccines has been highlighted by the rise of variants of concern (VoCs) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of the prefusion SARS-CoV-2 spike (S), S1, and receptor-binding domain (RBD). These immunogens induce robust S binding, ACE2 inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID 50 > 10,000) following a single immunization, whereas RBD-ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations and also show durable and potent neutralization against circulating VoCs. Passive transfer of immunoglobulin G (IgG) purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2-blocking activity and ID 50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens., Competing Interests: Declaration of interests M.G.J. and K.M. are named as inventors on international patent application WO/2021/178971 A1 entitled “Vaccines against SARS-CoV-2 and other coronaviruses.” M.G.J. is named as an inventor on international patent application WO/2018/081318 and U.S. patent 10,960,070 entitled “Prefusion coronavirus spike proteins and their use.” Z.B. is named as an inventor on U.S. patent 10,434,167 entitled “Non-toxic adjuvant formulation comprising a monophosphoryl lipid A (MPLA)-containing liposome composition and a saponin.” Z.B. and G.R.M. are named as inventors on U.S. patent application 16/607,917 entitled “Compositions and methods for vaccine delivery.” M.S.D. is a consultant for Inbios, Vir Biotechnology, Fortress Biotech, and Carnival Corporation and on the scientific advisory boards of Moderna and Immunome. The Diamond laboratory has received funding support from sponsored research agreements from Moderna, Vir Biotechnology, Kaleido, and Emergent BioSolutions. S.R., P.M.M., and M.T.E. are employees of AstraZeneca and currently hold AstraZeneca stock or stock options. Z.B. is currently employed at Pfizer., (Copyright © 2021. Published by Elsevier Inc.)

Plague is a rapidly lethal human disease caused by the bacterium Yersinia pestis This study demonstrated that the Y. pestis plasminogen activator Pla, a protease that promotes fibrin degradation, thwarts T cell-mediated defense against fully virulent Y. pestis Introducing a single point mutation into the active site of Pla suffices to render fully virulent Y. pestis susceptible to primed T cells. Mechanistic studies revealed essential roles for fibrin during T cell-mediated defense against Pla-mutant Y. pestis Moreover, the efficacy of T cell-mediated protection against various Y. pestis strains displayed an inverse relationship with their levels of Pla activity. Together, these data indicate that Pla functions to thwart fibrin-dependent T cell-mediated defense against plague. Other important human bacterial pathogens, including staphylococci, streptococci, and borrelia, likewise produce virulence factors that promote fibrin degradation. The discovery that Y. pestis thwarts T cell defense by promoting fibrinolysis suggests novel therapeutic approaches to amplifying T cell responses against human pathogens., (Copyright © 2019 American Society for Microbiology.)

Non-replicating vaccines, such as those based on recombinant proteins, require adjuvants and delivery systems, which have thus far depended on mimicking pathogen danger signals and strong pro-inflammatory responses. In search of a safer and more efficacious alternative, we tested whether vaccinations with influenza recombinant hemagglutinin (HA) mixed with a novel vegetable oil in water emulsion adjuvant (Natural Immune-enhancing Delivery System, NIDS), based on the immune-enhancing synergy of vitamins A and E and a catechin, could protect against intra-nasal challenge with live influenza virus. Vaccinations of inbred Brag Albino strain c (BALB/c) mice, with HA mixed with NIDS compared to other adjuvants, i.e., a squalene oil in water emulsion (Sq. oil), and the Toll Like Receptor 3 (TLR3) agonist Poly (I:C), induced significantly lower select innate pro-inflammatory responses in serum, but induced significantly higher adaptive antibody and splenic T Helper 1 (TH1) or TH2, but not TH17, responses. Vaccinations with NIDS protected against infection, as measured by clinical scores, lung viral loads, and serum hemagglutination inhibition titers. The NIDS exhibited a strong dose sparing effect and the adjuvant action of NIDS was intact in the outbred CD1 mice. Importantly, vaccinations with the Sq. oil, but not NIDS, induced a significantly higher Serum Amyloid P component, an acute phase reactant secreted by hepatocytes, and total serum IgE. Thus, the NIDS may be used as a clinically safer and more efficacious vaccine adjuvant against influenza, and potentially other infectious diseases., Competing Interests: Michael Vajdy owns shares of EpitoGenesis, Inc. Sapna Patel is a former employee of EpitoGenesis, Inc., with no shares in the company. All other authors were independent subcontractors or consultants with no conflicts of interest.

Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE69-77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE69-77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE69-77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE69-77-mediated protection. In contrast, YopE69-77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias.

Immunomodulatory agents potentially represent a new class of broad-spectrum antimicrobials. Here, we demonstrate that prophylaxis with immunomodulatory cytosine-phosphate-guanidine (CpG) oligodeoxynucleotide (ODN), a toll-like receptor 9 (TLR9) agonist, confers protection against Yersinia pestis, the etiologic agent of plague. The data establish that intranasal administration of CpG ODN 1 day prior to lethal pulmonary exposure to Y. pestis strain KIM D27 significantly improves survival of C57BL/6 mice and reduces bacterial growth in hepatic tissue, despite paradoxically increasing bacterial growth in the lung. All of these CpG ODN-mediated impacts, including the increased pulmonary burden, are TLR9 dependent, as they are not observed in TLR9-deficient mice. The capacity of prophylactic intranasal CpG ODN to enhance survival does not require adaptive immunity, as it is evident in mice lacking B and/or T cells; however, the presence of T cells improves long-term survival. The prophylactic regimen also improves survival and reduces hepatic bacterial burden in mice challenged intraperitoneally with KIM D27, indicating that intranasal delivery of CpG ODN has systemic impacts. Indeed, intranasal prophylaxis with CpG ODN provides significant protection against subcutaneous challenge with Y. pestis strain CO92 even though it fails to protect mice from intranasal challenge with that fully virulent strain.

The Gram-negative bacterium Yersinia pestis causes plague, a rapidly progressing and often fatal disease. The formation of fibrin at sites of Y. pestis infection supports innate host defense against plague, perhaps by providing a nondiffusible spatial cue that promotes the accumulation of inflammatory cells expressing fibrin-binding integrins. This report demonstrates that fibrin is an essential component of T cell-mediated defense against plague but can be dispensable for Ab-mediated defense. Genetic or pharmacologic depletion of fibrin abrogated innate and T cell-mediated defense in mice challenged intranasally with Y. pestis. The fibrin-deficient mice displayed reduced survival, increased bacterial burden, and exacerbated hemorrhagic pathology. They also showed fewer neutrophils within infected lung tissue and reduced neutrophil viability at sites of liver infection. Depletion of neutrophils from wild-type mice weakened T cell-mediated defense against plague. The data suggest that T cells combat plague in conjunction with neutrophils, which require help from fibrin to withstand Y. pestis encounters and effectively clear bacteria.

Influenza causes >250,000 deaths annually in the industrialized world, and bacterial infections frequently cause secondary illnesses during influenza outbreaks, including pneumonia, bronchitis, sinusitis, and otitis media. In this study, we demonstrate that cross-reactive immunity to mismatched influenza strains can reduce susceptibility to secondary bacterial infections, even though this fails to prevent influenza infection. Specifically, infecting mice with H3N2 influenza before challenging with mismatched H1N1 influenza reduces susceptibility to either Gram-positive Streptococcus pneumoniae or Gram-negative Klebsiella pneumoniae. Vaccinating mice with the highly conserved nucleoprotein of influenza also reduces H1N1-induced susceptibility to lethal bacterial infections. Both T cells and Abs contribute to defense against influenza-induced bacterial diseases; influenza cross-reactive T cells reduce viral titers, whereas Abs to nucleoprotein suppress induction of inflammation in the lung. These findings suggest that nonneutralizing influenza vaccines that fail to prevent influenza infection may nevertheless protect the public from secondary bacterial diseases when neutralizing vaccines are not available.

With age, peripheral naive CD4 T cells become both longer lived and functionally impaired and they express reduced levels of Bim, a proapoptotic Bcl family member. In this study, we show that reduced Bim expression by naive CD4 T cells intrinsically mediates their longer lifespan in the periphery. Moreover, using mixed bone marrow chimeras reconstituted with Bim(+/+) and Bim(+/-) bone marrow cells, Bim(+/-) naive CD4 T cells exhibit accelerated development of age-associated dysfunctions, including reduced proliferation and IL-2 production and defective helper function for B cells, without any increase in their turnover. However, newly generated Bim(+/-) naive CD4 T cells in middle-aged mice are not defective, indicating an additional requirement for their persistence in the periphery. These age-associated immune defects develop independently of the "aged" host environment and without extensive division, distinguishing them from classic "senescence." We suggest that the reduction of Bim levels with age in naive CD4 T cell is the initiating step that leads to increased cellular lifespan and development of age-associated functional defects.

With age, T-cell generation from the thymus is much reduced, yet a substantial naïve T-cell pool is maintained even in aged animals, suggesting that naïve T cells either persist longer or turn over faster to maintain T-cell homeostasis. We found that with age, naïve CD4 T cells became progressively longer-lived. Their longer lifespan did not depend on recognition of self-peptide/class II. Newly generated naïve T cells derived from aged stem cells had a shorter lifespan, like that of young naïve T cells. Conversely, naïve CD4 T cells derived from middle-aged thymectomized mice were longer-lived in vivo, and their development of functional defects was accelerated. These observations suggest that naïve T cells develop their longer lifespan during their sojourn in the periphery. Increased longevity of naïve CD4 T cells correlated well with reduced expression of proapoptotic molecule Bim. We suggest that the intrinsic increase in longevity helps maintain naïve T-cell homeostasis but facilitates the development of functional defects in mice.

Naive CD4 cells from aged mice respond inefficiently to Ag, but the factors that underlie the age-associated defects remain unclear. We have used two approaches to isolate recent thymic emigrants (RTE) in young and aged mice and have compared their capacity to respond to antigenic stimulation ex vivo. An in situ intrathymic CFSE injection labeled developing thymocytes and allowed the identification of RTE in secondary lymphoid tissues. Analysis of CFSE-labeled RTE and control unlabeled naive CD4 cells indicated that cells from aged mice were defective in their ability to increase intracellular Ca(2+) concentration following TCR cross-linking. Aged naive and RTE CD4 also secreted less IL-2 and proliferated less than that of comparable young CD4 populations. Defects in effector generation in aged RTE were overcome by the addition of IL-2 to cultures. RTE from both polyclonal and TCR transgenic mice were compromised, indicating that defects were independent of TCR specificity. In the second model, the cotransfer of congenic marker-labeled young and aged BM cells into young and aged syngeneic hosts revealed that hyporesponsiveness in aged RTE was caused by a combination of defects intrinsic to CD4 progenitors and defects induced by the aged environment. Depletion of peripheral CD4 cells in aged mice led to production of new RTE that were not defective. The results of this study suggest that defects induced by environmental and lineage intrinsic factors act together to reduce responses to Ag in aged naive CD4 cells and that these defects can be overcome in aged CD4 cells produced during recovery from lymphopenia.

Although B cells produce cytokines it is not known whether B cells can differentiate into effector subsets that secrete polarized arrays of cytokines. We have identified two populations of "effector" B cells (Be1 and Be2) that produce distinct patterns of cytokines depending on the cytokine environment in which the cells were stimulated during their primary encounter with antigen and T cells. These effector B cell subsets subsequently regulate the differentiation of naïve CD4+ T cells to TH1 and TH2 cells through production of polarizing cytokines such as interleukin 4 and interferon gamma. In addition, Be1 and Be2 cells could be identified in animals that were infected with pathogens that preferentially induce a Type 1 and Type 2 immune response. Together these results suggest that, in addition to their well defined role in antibody production, B cells may regulate immune responses to infectious pathogens through their production of cytokines.