The COVID-19 pandemic has accelerated interest in immuno-multiple reaction monitoring (immuno-MRM) for peptide quantification, with early efforts focusing on SARS-CoV-2 biomarker detection in clinical nasopharyngeal swabs. However, the emergence of mRNA vaccines has created a new and pressing need for robust methods to quantify antigen expression. Here, we present an optimized immuno-MRM method targeting the SARS-CoV-2 spike fusion peptide SFIEDLLFNK, designed and validated to quantify antigen expression following mRNA or plasmid transfection. This method offers high sensitivity, precision, and linearity across a broad dynamic range, enabling an accurate assessment of protein translation in vitro. In addition to measuring antigen levels, analysis of the flow-through provides insight into host cell proteomic responses, supporting the comprehensive characterization of mRNA vaccine efficacy and safety. This dual-function workflow serves as a powerful tool for vaccine development, quality control, and regulatory evaluation of RNA-based therapeutics.

ABSTRACT Severe COVID-19 is frequently associated with vascular complications, raising ongoing debate about whether SARS-CoV-2 can directly infect endothelial cells and thereby contribute to disease pathogenesis. Although endothelial cells express angiotensin-converting enzyme 2 (ACE2), the in vivo relevance of endothelial-restricted viral tropism remains unclear. To directly assess the consequences of endothelial-restricted SARS-CoV-2 tropism in vivo , we generated a transgenic mouse model expressing human ACE2 under control of the endothelial-specific Cdh5 promoter ( Cdh5 -hACE2). Despite confirmed pulmonary endothelial expression and protein presence of hACE2, SARS-CoV-2 infection of Cdh5 -hACE2 mice did not induce clinical illness, detectable viral replication, immune cell influx in the lung, or histopathological abnormalities in the lung or brain. These findings indicate that endothelial-restricted SARS-CoV-2 tropism alone is insufficient to drive productive infection and clinical disease in vivo , suggesting that endothelial involvement in COVID-19 likely arises in the context of broader cellular infection or systemic host responses rather than from primary endothelial infection. IMPORTANCE Although SARS-CoV-2 primarily infects the upper and lower airways, COVID-19 was quickly recognized as a multi-organ disease, in which vascular complications are a recurring feature. This has raised the possibility that direct infection of endothelial cells contributes to disease pathogenesis. However, whether vascular injury arises from productive endothelial infection or instead represents a secondary consequence of systemic inflammation remains unresolved. To directly disentangle these possibilities and define the in vivo consequences of endothelial-restricted viral tropism, we generated a transgenic mouse model expressing human ACE2 under the control of the endothelial-specific Cdh5 promoter ( Cdh5 -hACE2).

Abstract Background More than 7 million people have died from COVID-19 to date and SARS-CoV-2 continues to cause substantial disease around the world. High-risk patients like immunocompromised and elderly are often not able to elicit adequate immune responses to vaccination and strongly benefit from an additional layer of protection in the form of complementary antibody therapy. Monoclonal antibodies act immediately and can provide immune support for months. However, all previously authorized antibodies lost their neutralization potency against the currently circulating and constantly mutating SARS-CoV-2 variants, leaving a vast unmet medical need. The combination XVR012 consists of the molecules XVR013m and XVR014. Methods The combination XVR012 consists of the molecules XVR013m and XVR014 (Figure 1). XVR013m is a mono-specific VHH-Fc antibody that targets a unique epitope in the S2 subunit, XVR014 is a bi-specific bivalent VHHx-Fc-VHHy antibody construct, targeting 2 non-competing epitopes in the Receptor-Binding Domain of the spike (Figure 2). Both antibodies contain a human IgG1 Fc with LS mutations for half-life extension. Structural analysis of the XVR013m epitope Left panel: Structural analysis of the VHH R3DC23 (green) bound to the HR2 domain of the S2 unit of the SARS-CoV-2 spike protein, between the terminal N1194 glycan (orange) and the viral membrane. Right panel: One R3DC23 (green) binds the interface of two HR2 coils and each HR2 coil is bound by two R3DC23 VHHs. Results XVR012 delivers a triple mode of action in preventing infection of host cells, i.e., (1) sterical hinderance of ACE2 receptor binding, (2) induction of S1 subunit shedding thereby preventing viral attachment and (3) inhibition of the fusion process between the viral and host cell membrane. XVR012 broadly neutralizes SARS-CoV-1 and SARS-CoV-2 viruses in vitro and demonstrates an IC50 ranging from 4.8 to 8,7 ng/mL against all SARS-CoV-2 variants tested so far in a pseudovirus neutralization assay, including the currently circulating BA.2.86.1, HK.3, EG.5.1 and HV.1 (Table 1). In vivo, efficacy was demonstrated in the Syrian Golden Hamster model in a therapeutic setting, showing complete reduction of lung viremia for all XVR012 dose levels tested, even with a very low dose of 0,5 mg/kg for the XVR013m component. (Figure 3). PK parameters have been determined in a Tg32 SCID mouse model and support an envisioned duration of protection up to 6 months. Neutralization potency of VSV pseudo-typed with spike proteins of recent SARS-CoV-2 variants. Data are presented as mean IC50 based on 3 independent experiments, except for (*) 1 independent experiment. Conclusion In summary, this second-generation cocktail targeting three unique and highly conserved epitopes in the S1 and S2 subunits of the spike is ready to proceed to clinical testing and may provide a long-term solution to the populations at highest risk. Lung infectious viral titers in Syrian golden hamster post-infection challenge model (Wuhan strain) on day 4 post infection. Mean values + standard error of the mean (SEM) are reported. * Two animals in 2 mg/kg group of XVR013m and one animal in the 20 mg/kg group of XVR014 were experimentally confirmed in PK assays to not have been exposed to the drug. Dotted lines represent the lower limit of detection (LLOD) range. Disclosures Florence M. Herschke, PhD, ExeVir Bio: Patent inventor|ExeVir Bio: Employee Bert Schepens, PhD, Exevir: Bert Schepens is Inventor on patents that describe the nanobodies that are mentioned in the abstract and presentation Loes van Schie, PhD, Exevir: Grant/Research Support|Exevir: I am a co-inventor on the patents protecting the biologicals described in the abstract and presentation Xavier Saelens, PhD, ExeVir Bio: I am an inventor on patent applications WO2022/167666 A1 and WO2023/222825 A1 which incorporate discoveries and inventions described here.|ExeVir Bio: I am a scientific co-founder of ExeVir Bio and in receipt of ExeVir Bio share options

Therapeutic monoclonal antibodies can prevent severe disease in SARS-CoV-2 exposed individuals. However, currently circulating virus variants have evolved to gain significant resistance to nearly all neutralizing human immune system-derived therapeutic monoclonal antibodies that had previously been emergency-authorized for use in the clinic. Here, we describe the discovery of a panel of single-domain antibodies (VHHs) directed against the spike protein S2 subunit that broadly neutralize SARS-CoV-1 and -2 with unusually high potency. One of these VHHs tightly clamps the spike's monomers at a highly conserved, quaternary epitope in the membrane proximal part of the trimeric Heptad Repeat 2 (HR2) coiled-coil, thereby locking the HR2 in its prefusion conformation. Low dose systemic administration of a VHH-human IgG1 Fc fusion prevented SARS-CoV-2 infection in two animal models. Pseudovirus escape selection experiments demonstrate that the very rare escape variants are rendered almost non-infectious. This VHH-based antibody with a highly potent mechanism of antiviral action forms the basis for a new class of pan-sarbecovirus neutralizing biologics, which are currently under development. In addition, the unique quaternary binding mode of the VHHs to the prefusion HR2 could be exploited for other class I fusion proteins.

The COVID-19 pandemic has catalyzed interest in immuno-multiple reaction monitoring (immuno-MRM) methods, with the detection of peptides unique to the nucleocapsid protein in nasopharyngeal swabs. While current applications predominantly focus on disease biomarkers, the pandemic has unveiled new opportunities, namely for the quantification of antigen expression following mRNA vaccination. Here, we present an optimized immuno-MRM method for quantifying SARS-CoV-2 spike protein fusion peptide, SFIEDLLFNK, for several practical applications. The method is versatile, applicable to multiple biological matrices, including plasma, and can be extended to nasopharyngeal swabs. It also offers a high-precision tool for assessing protein expression following plasmid and mRNA transfection. Moreover, in parallel to enabling accurate antigen quantification, the flow-through can be used to determine the proteome profile of the infected cells, providing insights into the intracellular immune response. This dual capability supports the rapid optimization of mRNA vaccines, thereby driving advancements in vaccine development strategies. ### Competing Interest Statement The authors declare the following competing financial interest(s): Vanhulle M. and Vissers J.P.C. are employed by Waters Corporation.

Given the ongoing evolution of SARS-CoV-2 and the historical emergence of other highly transmissible coronaviruses like SARS-CoV and HCoV-NL63, in vivo coronavirus research remains crucial even when the COVID-19 pandemic is receding. Due to restricted tropism of SARS-CoV-2 and other coronaviruses for mouse cells, model systems of infection rely on transgenic expression of the entry receptor human ACE2 (hACE2). Available hACE2 transgenic models using the Krt18 promotor express the receptor across multiple cell types and organ systems, leading to multiple disease features including pneumonia, vascular compromise and neuro-inflammation, that reflect the multi-organ nature of COVID-19. To disentangle the role of cell tropism in driving the clinical manifestations of the disease, we generated two new transgenic mouse models, Sftpa1 -hACE2 and Cdh5 -hACE2 transgenic mice, with hACE2 restricted to lung epithelium or endothelial cells respectively. In Sftpa1 -hACE2 mice, with high expression of the hACE2 receptor in lung alveolar type 2 cells, SARS-CoV-2 infection led to rapidly progressing disease, characterised by a strong neutrophilic innate immune response in the lung, followed by viral neuro-invasion and early death. Krt18 -hACE2 mice additionally recruited various dendritic cell subsets and gradually developed adaptive immunity. In Cdh5 -hACE2 Tg mice with exclusive endothelial tropism of the virus, viral inoculation via the lung or systemic circulation did not lead to viral propagation or disease manifestations, despite endothelial expression of hACE2 in the lung. These results suggest that tropism for alveolar epithelial cells increases disease severity, while endothelial cell tropism per se does not drive the vascular consequences often seen in COVID-19 patients. Our new transgenic mouse models will be helpful to dissect how cell tropism contributes to the clinical manifestations of coronavirus infection. ### Competing Interest Statement The authors have declared no competing interest.

Monoclonal antibodies are the leading drug of the biopharmaceutical market because of their high specificity and tolerability, but the current CHO-based manufacturing platform remains expensive and time-consuming leading to limited accessibility, especially in the case of diseases with high incidence and pandemics. Therefore, there is an urgent need for an alternative production system.In this study, we present a rapid and cost-effective microbial platform for heavy chain-only antibodies (VHH-Fc) in the methylotrophic yeast Komagataella phaffii (aka Pichia pastoris ). We demonstrate the potential of this platform using a simplified single-gene VHH-Fc fusion construct instead of the conventional monoclonal antibody format, as this is more easily expressed in Pichia pastoris . We demonstrate that the Pichia -produced VHH-Fc fusion construct is stable and that a Pichia -produced VHH-Fc directed against the SARS-CoV-2 spike has potent SARS-CoV-2 neutralizing activity in vitro and in vivo . We expect that this platform will pave the way towards faster and cheaper development and production of broadly neutralizing single-chain antibodies in yeast.### Competing Interest StatementC.L., H.E., K.R., B.S., S.D.C., D.F., W.N., J.N., X.S., L.v.S., and N.C. are named as inventors on patent application Coronavirus Binders (WO 2021/156490 A2), published on 12 August 2021. C.L., B.S., W.W., L.v.S. and N.C. are named as inventors on patent application Engineered stabilizing aglycosylated fc-regions (WO 2023/148397 A1), published on 10 August 2023. X.S. and N.C. are scientific founders of and consultants for ExeVir Bio and are in receipt of ExeVir Bio share options. All other authors declare that they have no competing interests.

Background SARS-CoV-2-neutralizing antibodies (nABs) showed great promise in the early phases of the COVID-19 pandemic. The emergence of resistant strains, however, quickly rendered the majority of clinically approved nABs ineffective. This underscored the imperative to develop nAB cocktails targeting non -overlapping epitopes. Methods Undertaking a nAB discovery program, we employed a classical workflow, while integrating artificial intelligence (AI) -based prediction to select non -competing nABs very early in the pipeline. We identified and in vivo validated (in female Syrian hamsters) two highly potent nABs. Findings Despite the promising results, in depth cryo-EM structural analysis demonstrated that the AI -based prediction employed with the intention to ensure non -overlapping epitopes was inaccurate. The two nABs in fact bound to the same receptor -binding epitope in a remarkably similar manner. Interpretation Our findings indicate that, even in the Alphafold era, AI -based predictions of paratope-epitope interactions are rough and experimental validation of epitopes remains an essential cornerstone of a successful nAB lead selection. Copyright (c) 2023 Published by Elsevier B.V. This is an open access article under the CC BY -NC -ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

As small and stable high-affinity antigen binders, VHHs boast attractive characteristics both for therapeutic use in various disease indications, and as versatile reagents in research and diagnostics. To further increase the versatility of VHHs, we explored the VHH scaffold in a structure-guided approach to select regions where the introduction of an N-glycosylation N-X-T sequon and its associated glycan should not interfere with protein folding or epitope recognition. We expressed variants of such glycoengineered VHHs in the Pichia pastoris GlycoSwitchM5 strain, allowing us to pinpoint preferred sites at which Man 5 GlcNAc 2 -glycans can be introduced at high site occupancy without affecting antigen binding. A VHH carrying predominantly a Man 5 GlcNAc 2 N-glycan at one of these preferred sites showed highly efficient, glycan-dependent uptake by Mf4/4 macrophages in vitro and by alveolar lung macrophages in vivo, illustrating one potential application of glyco-engineered VHHs: a glycan-based targeting approach for lung macrophage endolysosomal system delivery. The set of optimal artificial VHH N-glycosylation sites identified in this study can serve as a blueprint for targeted glyco-engineering of other VHHs, enabling site-specific functionalization through the rapidly expanding toolbox of synthetic glycobiology.

Currently circulating SARS-CoV-2 variants have gained complete or significant resistance to all SARS-CoV-2-neutralizing antibodies that have been used in the clinic. Such antibodies can prevent severe disease in SARS-CoV-2 exposed patients for whom vaccines may not provide optimal protection. Here, we describe single-domain antibodies (VHHs), also known as nanobodies, that can broadly neutralize SARS-CoV-2 with unusually high potency. Structural analysis revealed their binding to a unique, highly conserved, membrane proximal, quaternary epitope in the S2 subunit of the spike. Furthermore, a VHH-human IgG1 Fc fusion, efficiently expressed in Chinese hamster ovary cells as a stable antibody construct, protected hamsters against SARS-CoV-2 replication in a therapeutic setting when administered systemically at low dose. This VHH-based antibody represents a new candidate anti-COVID-19 biologic that targets the Achilles heel of the viral spike.

Respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Although several pattern recognition receptors (PRRs) can sense RSV-derived pathogen-associated molecular patterns (PAMPs), infection with RSV is typically associated with low to undetectable levels of type I interferons (IFNs). Multiple RSV proteins can hinder the host's innate immune response. The main players are NS1 and NS2 which suppress type I IFN production and signalling in multiple ways. The recruitment of innate immune cells and the production of several cytokines are reduced by RSV G. Next, RSV N can sequester immunostimulatory proteins to inclusion bodies (IBs). N might also facilitate the assembly of a multiprotein complex that is responsible for the negative regulation of innate immune pathways. Furthermore, RSV M modulates the host's innate immune response. The nuclear accumulation of RSV M has been linked to an impaired host gene transcription, in particular for nuclear-encoded mitochondrial proteins. In addition, RSV M might also directly target mitochondrial proteins which results in a reduced mitochondrion-mediated innate immune recognition of RSV. Lastly, RSV SH might prolong the viral replication in infected cells and influence cytokine production.

RIPK3 partially protects against disease caused by influenza A virus (IAV) infection in the mouse model. Here, we compared the immune protection of active vaccination with a universal influenza A vaccine candidate based on the matrix protein 2 ectodomain (M2e) and of passive immunization with anti-M2e IgG antibodies in wild type and Ripk3 −/− mice. We observed that the protection against IAV after active vaccination with M2e viral antigen is lost in Ripk3 −/− mice. Interestingly, M2e-specific serum IgG levels induced by M2e vaccination were not significantly different between wild type and Ripk3 −/− vaccinated mice demonstrating that the at least the humoral immune response was not affected by the absence of RIPK3 during active vaccination. Moreover, following IAV challenge, lungs of M2e vaccinated Ripk3 − /− mice revealed a decreased number of immune cell infiltrates and an increased accumulation of dead cells, suggesting that phagocytosis could be reduced in Ripk3 −/− mice. However, neither efferocytosis nor antibody-dependent phagocytosis were affected in macrophages isolated from Ripk3 − /− mice. Likewise following IAV infection of Ripk3 −/− mice, active vaccination and infection resulted in decreased presence of CD8+ T-cells in the lung. However, it is unclear whether this reflects a deficiency in vaccination or an inability following infection. Finally, passively transferred anti-M2e monoclonal antibodies at higher dose than littermate wild type mice completely protected Ripk3 −/− mice against an otherwise lethal IAV infection, demonstrating that the increased sensitivity of Ripk3 −/− mice could be overcome by increased antibodies. Therefore we conclude that passive immunization strategies with monoclonal antibody could be useful for individuals with reduced IAV vaccine efficacy or increased IAV sensitivity, such as may be expected in patients treated with future anti-inflammatory therapeutics for chronic inflammatory diseases such as RIPK inhibitors.

Innate immune responses, including the production of type I and III interferons, play a crucial role in the first line of defense against RSV infection. However, only a poor induction of type I IFNs is observed during RSV infection, suggesting that RSV has evolved mechanisms to prevent type I IFN expression by the infected host cell.

A double hit with one antibody construct may avoid viral escape

The ectodomain of matrix protein 2 (M2e) of influenza A viruses is a universal influenza A vaccine candidate. Here, we report potential evasion strategies of influenza A viruses under in vivo passive anti-M2e IgG immune selection pressure in severe combined immune-deficient (SCID) mice. A/Puerto Rico/8/34-infected SCID mice were treated with the M2e-specific mouse IgG monoclonal antibodies (MAbs) MAb 65 (IgG2a) or MAb 37 (IgG1), which recognize amino acids 5 to 15 in M2e, or with MAb 148 (IgG1), which binds to the invariant N terminus of M2e. Treatment of challenged SCID mice with any of these MAbs significantly prolonged survival compared to isotype control IgG treatment. Furthermore, M2e-specific IgG2a protected significantly better than IgG1, and even resulted in virus clearance in some of the SCID mice. Deep sequencing analysis of viral RNA isolated at different time points after treatment revealed that the sequence variation in M2e was limited to P10H/L and/or I11T in anti-M2e MAb-treated mice. Remarkably, in half of the samples isolated from moribund MAb 37-treated mice and in all MAb 148-treated mice, virus was isolated with a wild-type M2 sequence but with nonsynonymous mutations in the polymerases and/or the hemagglutinin genes. Some of these mutations were associated with delayed M2 and other viral gene expression and with increased resistance to anti-M2e MAb treatment of SCID mice. Treatment with M2e-specific MAbs thus selects for viruses with limited variation in M2e. Importantly, influenza A viruses may also undergo an alternative escape route by acquiring mutations that result in delayed wild-type M2 expression. IMPORTANCE Broadly protective influenza vaccine candidates may have a higher barrier to immune evasion compared to conventional influenza vaccines. We used Illumina MiSeq deep sequence analysis to study the mutational patterns in A/Puerto Rico/8/34 viruses that evolve in chronically infected SCID mice that were treated with different M2e-specific MAbs. We show that under these circumstances, viruses emerged in vivo with mutations in M2e that were limited to positions 10 and 11. Moreover, we discovered an alternative route for anti-M2e antibody immune escape, in which a virus is selected with wild-type M2e but with mutations in other gene segments that result in delayed M2 and other viral protein expression. Delayed expression of the viral antigen that is targeted by a protective antibody thus represents an influenza virus immune escape mechanism that does not involve epitope alterations.

We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and −2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and −2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract disease, especially in young children and the elderly. The fusion protein (F) exists in a pre- and postfusion conformation and is the main target of RSV-neutralizing antibodies. Highly potent RSV-neutralizing antibodies typically bind sites that are unique to the prefusion conformation of F. In this study we screened a single-domain antibody (VHH) library derived from a llama immunized with prefusion-stabilized F and identified a prefusion F-specific VHH that can neutralize RSV A at subnanomolar concentrations. Structural analysis revealed that this VHH primarily binds to antigenic site I while also making contacts with residues in antigenic site III and IV. This new VHH reveals a previously underappreciated membrane-proximal region sensitive for neutralization.ImportanceRSV is an important respiratory pathogen. This study describes a prefusion F-specific VHH that primarily binds to antigenic site I of RSV F. This is the first time that a prefusion F-specific antibody that binds this site is reported. In general, antibodies that bind to site I are poorly neutralizing, whereas the VHH described here neutralizes RSV A at subnanomolar concentrations. Our findings contribute to insights into the RSV F antigenic map.

Broadly neutralizing antibodies are an important treatment for individuals with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antibody-based therapeutics are also essential for pandemic preparedness against future Sarbecovirus outbreaks. Camelid-derived single domain antibodies (VHHs) exhibit potent antimicrobial activity and are being developed as SARS-CoV-2–neutralizing antibody-like therapeutics. Here, we identified VHHs that neutralize both SARS-CoV-1 and SARS-CoV-2, including now circulating variants. We observed that the VHHs bound to a highly conserved epitope in the receptor binding domain of the viral spike protein that is difficult to access for human antibodies. Structure-guided molecular modeling, combined with rapid yeast-based prototyping, resulted in an affinity enhanced VHH-human immunoglobulin G1 Fc fusion molecule with subnanomolar neutralizing activity. This VHH-Fc fusion protein, produced in and purified from cultured Chinese hamster ovary cells, controlled SARS-CoV-2 replication in prophylactic and therapeutic settings in mice expressing human angiotensin converting enzyme 2 and in hamsters infected with SARS-CoV-2. These data led to affinity-enhanced selection of the VHH, XVR011, a stable anti–COVID-19 biologic that is now being evaluated in the clinic.

The small hydrophobic (SH) glycoprotein of human respiratory syncytial virus (RSV) is a transmembrane protein that is poorly accessible by antibodies on the virion but has an ectodomain (SHe) that is accessible and expressed on infected cells. The SHe from RSV strain A has been formulated in DPX, a unique delivery platform containing an adjuvant, and is being evaluated as an RSV vaccine candidate. The proposed mechanism of protection is the immune-mediated clearance of infected cells rather than neutralization of the virion. Our phase I clinical trial data clearly showed that vaccination resulted in robust antibody responses, but it was unclear if these immune responses have any correlation to immune responses to natural infection with RSV. Therefore, we embarked on this study to examine these immune responses in older adults with confirmed RSV infection. We compared vaccine-induced (DPX-RSV(A)) immune responses from participants in a Phase 1 clinical trial to paired acute and convalescent titers from older adults with symptomatic laboratory-confirmed RSV infection. Serum samples were tested for anti-SHe IgG titers and the isotypes determined. T cell responses were evaluated by IFN-γ ELISPOT. Anti-SHe titers were detected in 8 of 42 (19%) in the acute phase and 16 of 42 (38%) of convalescent serum samples. IgG1, IgG3, and IgA were the prevalent isotypes generated by both vaccination and infection. Antigen-specific T cell responses were detected in 9 of 16 (56%) of vaccinated participants. Depletion of CD4+ but not CD8+ T cells abrogated the IFN-γ ELISPOT response supporting the involvement of CD4+ T cells in the immune response to vaccination. The data showed that an immune response like that induced by DPX-RSV(A) could be seen in a subset of participants with confirmed RSV infection. These findings show that older adults with clinically significant infection as well as vaccinated adults generate a humoral response to SHe. The induction of both SHe-specific antibody and cellular responses support further clinical development of the DPX-RSV(A) vaccine.