Characterization of prefusion-F-specific antibodies elicited by natural infection with human metapneumovirus.

Human metapneumovirus (hMPV) is a major cause of acute respiratory infections in infants and older adults, for which no vaccines or therapeutics are available. The viral fusion (F) glycoprotein is required for entry and is the primary target of neutralizing antibodies; however, little is known about the humoral immune response generated from natural infection. Here, using prefusion-stabilized F proteins to interrogate memory B cells from two older adults, we obtain over 700 paired non-IgM antibody sequences representing 563 clonotypes, indicative of a highly polyclonal response. Characterization of 136 monoclonal antibodies reveals broad recognition of the protein surface, with potently neutralizing antibodies targeting each antigenic site. Cryo-EM studies further reveal two non-canonical sites and the molecular basis for recognition of the apex of hMPV F by two prefusion-specific neutralizing antibodies. Collectively, these results provide insight into the humoral response to hMPV infection in older adults and will help guide vaccine development.

Development of multivalent mRNA vaccine candidates for seasonal or pandemic influenza.

Recent approval of mRNA vaccines for emergency use against COVID-19 is likely to promote rapid development of mRNA-based vaccines targeting a wide range of infectious diseases. Compared to conventional approaches, this vaccine modality promises comparable potency while substantially accelerating the pace of development and deployment of vaccine doses. Already demonstrated successfully for single antigen vaccines such as for COVID-19, this technology could be optimized for complex multi-antigen vaccines. Herein, utilizing multiple influenza antigens, we demonstrated the suitability of the mRNA therapeutic (MRT) platform for such applications. Seasonal influenza vaccines have three or four hemagglutinin (HA) antigens of different viral subtypes. In addition, influenza neuraminidase (NA), a tetrameric membrane protein, is identified as an antigen that has been linked to protective immunity against severe viral disease. We detail the efforts in optimizing formulations of influenza candidates that use unmodified mRNA encoding full-length HA or full-length NA encapsulated in lipid nanoparticles (LNPs). HA and NA mRNA-LNP formulations, either as monovalent or as multivalent vaccines, induced strong functional antibody and cellular responses in non-human primates and such antigen-specific antibody responses were associated with protective efficacy against viral challenge in mice.

Effect of genetic background and delivery route on the preclinical properties of a live attenuated RSV vaccine.

A safe and effective vaccine against RSV remains an important unmet public health need. Intranasally (IN) delivered live-attenuated vaccines represent the most extensively studied approach for immunization of RSV-naïve infants and children, however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we report pre-clinical immunogenicity and efficacy data utilizing a live-attenuated vaccine candidate, RGΔM2-2, which was obtained by deleting the M2-2 open reading frame from the genome of the MSA1 clinical isolate. Intramuscular (IM) administration of RGΔM2-2 in cotton rats induced immunity and protective efficacy that was comparable to that induced by intranasal (IN) immunization. In contrast, the protective efficacy of RGΔM2-2 delivered by the IM route to African green monkeys was substantially reduced as compared to the efficacy following IN administration, despite comparable levels of serum neutralizing antibodies. This result suggests that mucosal immunity may play an important role in RSV protection. The RGΔM2-2 vaccine also demonstrated different attenuation profiles when tested in cotton rats, non-human primates, and a human airway epithelial (HAE) cell model. The data suggest RGΔM2-2 is less attenuated than a similarly designed vaccine candidate constructed on the A2 genetic background. These findings have important implications with regard to both the design and the preclinical safety testing of live-attenuated vaccines.

Core bead chromatography for preparation of highly pure, infectious respiratory syncytial virus in the negative purification mode.

Respiratory syncytial virus (RSV) is an important human pathogen, and is the most frequent viral cause of severe respiratory disease in infants. In addition, it is increasingly being recognized as an important cause of respiratory disease in the elderly and immunocompromised. Although a passive prophylactic treatment does exist for high-risk neonates and children, the overall disease burden warrants the development of a safe and effective prophylactic vaccine for use in otherwise healthy newborns and children. RSV is known to be an extremely labile virus, prone to aggregation and loss of infectious titer during virus handling and preparation procedures. To date infective RSV virions have been prepared by methods which are not readily scalable, such as density gradient ultracentrifugation. In this study we describe a scalable, chromatography-based purification procedure for preparation of highly pure, infectious RSV. The purification scheme is based on core bead technology and hollow fiber tangential flow filtration (TFF) and results in a ∼60% recovery of infectious virus titer. This method can be used to prepare highly purified wild type or live-attenuated vaccine strain viruses with titers as high as 1×10(8) plaque forming units per mL. A live-attenuated RSV vaccine prepared by this method was found to be immunogenic and protective in vivo, and its purity was 50-200-fold greater with respect to host cell dsDNA and Vero host cell proteins, than the raw feed stream. The results presented here can be considered a starting point for downstream process development of a live-attenuated vaccine approach for prevention of disease by RSV.

CX3CR1 Is Expressed in Differentiated Human Ciliated Airway Cells and Co-Localizes with Respiratory Syncytial Virus on Cilia in a G Protein-Dependent Manner.

Respiratory syncytial virus (RSV) is the principal cause of bronchiolitis in infants and a significant healthcare problem. The RSV Glycoprotein (G) mediates attachment of the virus to the cell membrane, which facilitates interaction of the RSV Fusion (F) protein with nucleolin, thereby triggering fusion of the viral and cellular membranes. However, a host protein ligand for G has not yet been identified. Here we show that CX3CR1 is expressed in the motile cilia of differentiated human airway epithelial (HAE) cells, and that CX3CR1 co-localizes with RSV particles. Upon infection, the distribution of CX3CR1 in these cells is significantly altered. Complete or partial deletion of RSV G results in viruses binding at least 72-fold less efficiently to cells, and reduces virus replication. Moreover, an antibody targeting an epitope near the G protein's CX3CR1-binding motif significantly inhibits binding of the virus to airway cells. Given previously published evidence of the interaction of G with CX3CR1 in human lymphocytes, these findings suggest a role for G in the interaction of RSV with ciliated lung cells. This interpretation is consistent with past studies showing a protective benefit in immunizing against G in animal models of RSV infection, and would support targeting the CX3CR1-G protein interaction for prophylaxis or therapy. CX3CR1 expression in lung epithelial cells may also have implications for other respiratory diseases such as asthma.

CPEB control of NF-kappaB nuclear localization and interleukin-6 production mediates cellular senescence.

CPEB is a sequence-specific translational regulatory RNA binding protein that mediates cellular senescence in primary mouse and human cells. CPEB knockout mouse embryo fibroblasts (MEFs) bypass senescence and synthesize large amounts of interleukin-6 (IL-6) and many other cytokines, which is not the case with either wild-type MEFs immortalized by prolonged culture or p53-deficient MEFs. CPEB regulates the production of IL-6 at both the translational and transcriptional levels; in CPEB-depleted cells, aberrant IL-6 transcription is mediated by improper NF-κB p65 phosphorylation and nuclear localization. Although IL-6 strengthens the senescence of wild-type cells, it has no effect on CPEB-deficient cells, even though they produce prodigious amounts of the cytokine. IL-6-promoted entry into senescence requires p53; CPEB knockout MEFs, however, synthesize only ∼50% of the p53 of wild-type MEFs, which is insufficient to respond to IL-6. Thus, CPEB deficiency not only increases IL-6 production but also renders the cell incapable of a senescence-promoting response.

Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site.

Cardioviruses have a unique 2A protein (143 aa). During genome translation, the encephalomyocarditis virus (EMCV) 2A is released through a ribosome skipping event mitigated through C-terminal 2A sequences and by subsequent N-terminal reaction with viral 3C(pro). Although viral replication is cytoplasmic, mature 2A accumulates in nucleoli shortly after infection. Some protein also transiently associates with cytoplasmic 40S ribosomal subunits, an activity contributing to inhibition of cellular cap-dependent translation. Cardiovirus sequences predict an eIF4E binding site (aa 126-134) and a nuclear localization signal (NLS, aa 91-102), within 2A, both of which are functional during EMCV infection. Point mutations preventing eIF4E:2A interactions gave small-plaque phenotype viruses, but still inhibited cellular cap-dependent translation. Deletions within the NLS motif relocalized 2A to the cytoplasm and abrogated the inhibition of cap-dependent translation. A fusion protein linking the 2A NLS to eGFP was sufficient to redirect the reporter to the nucleus but not into nucleoli.

Translational control from head to tail.

mRNA translation, a highly coordinated affair involving many proteins and RNAs, is generally divided into three steps: initiation, elongation, and termination. Each of these steps serves as a point of regulation to control the amount of protein that is produced. The protein 4E-HP has recently been shown to disrupt recruitment of the translation initiation complex by directly binding the 5' cap of cellular mRNAs. Recent work has shown elongation rates are probably altered during mitosis and certain types of synaptic transmission. Other work has shown premature termination of mRNAs lacking stop codons appears to repress their translation. Together, these studies highlight the importance of translational control in diverse processes such as development, cancer, and synaptic plasticity.

Cardiovirus 2A protein associates with 40S but not 80S ribosome subunits during infection.

Host translation shutoff induced in picornavirus-infected cells is a well-known phenomenon. The mechanisms by which separate genera of the picornavirus family achieve this shutoff differ. This study examined alterations in the cellular translational components in HeLa cells infected with encephalomyocarditis virus (EMCV), a cardiovirus. In agreement with previous reports, EMCV induced a marked decrease in host mRNA translation. The inhibition correlated with the appearance of a significantly enhanced 80S peak in cells and a concomitant decrease in polysome abundance. Characterization of the 80S material revealed that these ribosomes were virtually devoid of mRNA. Viral protein 2A was tightly associated with some of the free 40S ribosome subunits, but it was not present in the 80S pool which accumulated after infection. Expression of 2A protein in cells in the absence infection was able to modulate the cellular translational environment to increase the ratio of internal ribosome entry site-dependent translation to cap-dependent translation of a reporter construct. The results provide further evidence for a role of 2A protein in the mechanism of cardiovirus-induced host translational shutoff.