Pandemic Influenza Virus Research Articles

Innate immune control of influenza virus interspecies adaptation via IFITM3

Influenza virus pandemics are caused by viruses from animal reservoirs that adapt to efficiently infect and replicate in human hosts. Here, we investigate whether Interferon-Induced Transmembrane Protein 3 (IFITM3), a host antiviral factor with known human deficiencies, plays a role in interspecies virus infection and adaptation. We find that IFITM3-deficient mice and human cells can be infected with low doses of avian influenza viruses that fail to infect WT counterparts, identifying a new role for IFITM3 in controlling the minimum infectious virus dose threshold. Remarkably, influenza viruses passaged through Ifitm3−/− mice exhibit enhanced host adaptation, a result that is distinct from viruses passaged in mice deficient for interferon signaling, which exhibit attenuation. Our data demonstrate that IFITM3 deficiency uniquely facilitates potentially zoonotic influenza virus infections and subsequent adaptation, implicating IFITM3 deficiencies in the human population as a vulnerability for emergence of new pandemic viruses.

Pre-existing H1N1 immunity reduces severe disease with bovine H5N1 influenza virus.

The emergence of highly pathogenic H5N1 avian influenza in dairy cattle herds across the United States has caused multiple mild human infections. There is an urgent need to understand the risk of spillover into humans. Here, we show that pre-existing immunity from the 2009 H1N1 pandemic influenza virus provided protection from mortality and severe clinical disease to ferrets intranasally infected with bovine H5N1. H1N1 immune ferrets exhibited a differential tissue tropism with little bovine H5N1 viral dissemination to organs outside the respiratory tract and significantly less H5N1 virus found in nasal secretions and the respiratory tract. Additionally, ferrets with H1N1 prior immunity produced antibodies that cross-reacted with H5N1 neuraminidase protein. Taken together, these results suggest that mild disease in humans may be linked to prior immunity to human seasonal influenza viruses.

Unraveling the Role of Viral Interference in Disrupting Biennial RSV Epidemics in Northern Stockholm.

Respiratory syncytial virus (RSV) primarily impacts infants and older adults, with seasonal winter outbreaks in temperate countries. Biennial cycles of RSV activity have also been identified in Northern Europe and some states in the United States. Delayed RSV activity was reported worldwide during the 2009 influenza pandemic, and a disrupted biennial pattern of RSV activity was observed in northern Stockholm following the pandemic. Biennial patterns shifted to early/large outbreaks in even-numbered years and late/small outbreaks in odd-numbered years. However, the mechanisms underpinning this change in pattern remain unknown. In this work, we constructed an age-stratified mechanistic model to explicitly test three factors that could lead to the change in RSV transmission dynamics: 1) birth rates, 2) temperatures, and 3) viral interference. By fitting the model to weekly RSV admission data over a 20-year period and comparing different models, we found that viral interference from influenza was the only mechanism that explained the shifted biennial pattern. Our work demonstrates the complex interplay between different respiratory viruses, providing evidence that supports the presence of interactions between the H1N1 pandemic influenza virus and RSV at the population level, with implications for future public health interventions.

Gene editing of pigs to control influenza A virus infections

ABSTRACT Proteolytic activation of the haemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro. IAV infection in vivo resulted in a significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to wild-type pigs. Our findings support the commercial use of GE pigs to mitigate influenza A virus infection in pigs, as an alternative approach to prevent zoonotic influenza A transmissions from pigs to humans.

Cytomegalovirus vaccine vector-induced effector memory CD4 + T cells protect cynomolgus macaques from lethal aerosolized heterologous avian influenza challenge

An influenza vaccine approach that overcomes the problem of viral sequence diversity and provides long-lived heterosubtypic protection is urgently needed to protect against pandemic influenza viruses. Here, to determine if lung-resident effector memory T cells induced by cytomegalovirus (CMV)-vectored vaccines expressing conserved internal influenza antigens could protect against lethal influenza challenge, we immunize Mauritian cynomolgus macaques (MCM) with cynomolgus CMV (CyCMV) vaccines expressing H1N1 1918 influenza M1, NP, and PB1 antigens (CyCMV/Flu), and challenge with heterologous, aerosolized avian H5N1 influenza. All six unvaccinated MCM died by seven days post infection with acute respiratory distress, while 54.5% (6/11) CyCMV/Flu-vaccinated MCM survived. Survival correlates with the magnitude of lung-resident influenza-specific CD4 + T cells prior to challenge. These data demonstrate that CD4 + T cells targeting conserved internal influenza proteins can protect against highly pathogenic heterologous influenza challenge and support further exploration of effector memory T cell-based vaccines for universal influenza vaccine development.

Learning from the COVID-19 Pandemic: Next-generation universal vaccines and immunotherapeutic research

Learning from the COVID-19 Pandemic: Next-generation universal vaccines and immunotherapeutic research With the COVID-19 pandemic behind us, we need to focus on universal vaccines and/or immunotherapeutic strategies and technologies to tackle ongoing endemic infections with SARS-CoV2, influenza, and RSV and prepare for any future pandemics, says Dr Babita Agrawal. In the 21st century, we have witnessed the emergence of respiratory infections with pandemic potential, like corona and influenza viruses, on multiple occasions. Due to the global dissemination of one such coronavirus, SARS-CoV2 (severe acute respiratory syndrome-coronavirus type-2), the World Health Organization (WHO) declared a worldwide pandemic in March 2020. The global public health emergency was declared over in May 2023 by the WHO, but infections with variants of SARS-CoV2 continue to evolve and cause infections worldwide. (1) Besides public health measures, developing, approving, and implementing vaccines against SARS-CoV2 have helped mitigate and end the pandemic. However, the existing vaccines against SARS-CoV2 are not preventive, do not induce mucosal immunity, induce only short-term protection and are ineffective against emerging variants, thereby requiring regular updated boosters.

A Self-Fulfilling Prophecy Pandemic: The 1977 "Russian Flu".

Surprisingly, the 1977 "Russian flu" H1N1 pandemic influenza virus was genetically indistinguishable from strains that had circulated decades earlier but had gone extinct in 1957. This essay puts forward the most plausible chronology to explain the reemergence of the 1977 H1N1 pandemic virus: (1) in January-February 1976, a self-limited small outbreak of a swine H1N1 influenza virus occurred among Army personnel at Fort Dix, New Jersey; (2) in March 1976, the US launched a nationwide H1N1 swine influenza vaccine program; (3) other countries then also launched their own H1N1 R&D efforts; (4) a new H1N1 outbreak, genetically unrelated to the Fort Dix swine virus but indistinguishable from previously extinct H1N1 viruses, was detected early in 1977 in China; (5) the leading Chinese influenza virologist later disclosed that the Chinese military had conducted large H1N1 vaccine R&D studies in 1976. It is likely that the resurrected H1N1 influenza viruses were laboratory-stored strains that were unfrozen and studied as part of the emergency response to a perceived epidemic threat, and that accidentally escaped. The fear of a possible H1N1 pandemic was the critical factor that gave rise to the actual H1N1 pandemic, resulting in an avoidable "self-fulfilling prophecy pandemic."

Influenza H5N1 and H1N1 viruses remain infectious in unpasteurized milk on milking machinery surfaces.

Spillover of highly pathogenic avian H5N1 into the cattle population poses a risk to humans through the close contact with farm workers. High viral loads of influenza viruses in the unpasteurized milk of infected lactating cows has the potential to contaminate equipment within milking parlors and create fomites for transmission to dairy workers. Cattle H5N1 and human 2009 H1N1 pandemic influenza viruses were found to remain infectious on surfaces commonly found in milking equipment materials for a few hours. The data presented here provide a compelling case for the risk of contaminated surfaces generated during milking to facilitate transmission of H5N1 from cattle-to-cattle and to dairy farm workers.

Influenza antibody breadth and effector functions are immune correlates from acquisition of pandemic infection of children

Cross-reactive antibodies with Fc receptor (FcR) effector functions may mitigate pandemic virus impact in the absence of neutralizing antibodies. In this exploratory study, we use serum from a randomized placebo-controlled trial of seasonal trivalent influenza vaccination in children (NCT00792051) conducted at the onset of the 2009 H1N1 pandemic (pH1N1) and monitored for infection. We found that seasonal vaccination increases pH1N1 specific antibodies and FcR effector functions. Furthermore, prospective baseline antibody profiles after seasonal vaccination, prior to pH1N1 infection, show that unvaccinated uninfected children have elevated ADCC effector function, FcγR3a and FcγR2a binding antibodies to multiple pH1N1 proteins, past seasonal and avian (H5, H7 and H9) strains. Whereas, children that became pH1N1 infected after seasonal vaccination have antibodies focussed to seasonal strains without FcR functions, and greater aggregated HA-specific profiles for IgM and IgG3. Modeling to predict infection susceptibility, ranked baseline hemagglutination antibody inhibition as the highest contributor to lack of pH1N1 infection, in combination with features that include pH1-IgG1, H1-stem responses and FcR binding to seasonal vaccine and pH1 proteins. Thus, seasonal vaccination can have benefits against pandemic influenza viruses, and some children already have broadly reactive antibodies with Fc potential without vaccination and may be considered ‘elite influenza controllers’.

Preparation and characterization of curdlan-chitosan conjugate nanoparticles as mucosal adjuvants for intranasal influenza H1N1 subunit vaccine

Intranasal vaccination offers crucial protection against influenza virus pandemics. However, antigens, especially subunit antigens, often fail to induce effective immune responses without the help of immune adjuvants. Our research has demonstrated that a polyelectrolyte complex, composed of curdlan sulfate/O-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (CS/O-HTCC), effectively triggers both mucosal and systemic immune responses when administrated intranasal. In this study, stable nanoparticles formed by curdlan-O-HTCC conjugate (CO NP) were prepared and characterized. Furthermore, the efficacy of CO NP was evaluated as a mucosal adjuvant in an intranasal influenza H1N1 subunit vaccine. The results revealed that CO NP exhibits uniform and spherical morphology, with a size of 190.53 ± 4.22 nm, and notably, it remains stable in PBS at 4 °C for up to 6 weeks. Biological evaluation demonstrated that CO NP stimulates the activation of antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), both in vitro and in vivo. Furthermore, intranasal administration of CO NP effectively elicits cellular and humoral immune responses, notably enhancing mucosal immunity. Thus, CO NP emerges as a promising mucosal adjuvant for influenza subunit vaccines.

The Use of Neuraminidase Inhibitors in Teenagers May Not Increase the Risk of Neuropsychiatric Adverse Events: A Nationwide Population-based Retrospective Study

Background: Pandemic influenza virus is a public health issue, and the neuraminidase inhibitors (NIs) “Oseltamivir” and “Zanamivir” are effective treatments. While teenagers use NIs, there are concerns regarding neuropsychiatric adverse events (NPAEs). Aim: We aimed to use the Taiwan National Health Insurance Research Database to identify the correlation between NPAEs and NIs use in teenagers aged 13–19 years. Methods: The final population between 2000 and 2015 included in this study was 3698 individuals, with 2287 individuals having received “Oseltamivir” and “Zanamivir” (study cohort group) and 9148 individuals not receiving “Oseltamivir” and “Zanamivir” (comparison cohort group). We initially used a multivariate Cox regression analysis during the tracking period to determine the cumulative incidence of NPAEs. Results: Our findings revealed no significant increase in the likelihood of developing NPAEs in the study group. The Kaplan–Meier survival curve demonstrated that individuals who received “Oseltamivir” and “Zanamivir” were not associated with statistically significantly increased NPAEs compared with controls (log-rank test, P = 0.724). Conclusion: No more risk in comparison of the normal population in our study, and the safety of “Oseltamivir” and “Zanamivir” is established treatments for influenza.

Retraction: Properly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus.

Retraction: Properly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus.

Pandemic H1N1 influenza virus triggers a strong T helper cell response in human nasopharynx-associated lymphoid tissues

The pH1N1 belongs to influenza A family that is sometimes transmitted to humans via contact with pigs. Human tonsillar immune cells are widely used as in vitro models to study responses to influenza viruses. In the current study, human memory (M) and naïve (N) T cells responses in mononuclear cells of tonsil (TMCs) and peripheral blood (PBMCs) were stimulated by pH1N1/sH1N1, and then stained for estimation of T cells proliferation index. Individuals with an anti-pH1N1 hemagglutination (HA) inhibition (HAI) titer of forty or greater exhibited stronger HA-specific M-CD4+ T cells responses to pH1N1 in TMCs/PBMCs than those with an HAI titer of less than forty (P < 0.01). In addition, a positive correlation was observed between proliferation indices of M-CD4+ T cells induced by exposure to sH1N1/pH1N1 (p < 0.01). Moreover, a strong correlation (p < 0.001) was detected between subjects’ age and their HA-specific M-CD4+ T cells induced by pH1N1 exposure, indicating that this response was age-dependent. Finally, stimulation of TMCs with pH1N1-HA resulted in a significant M−CD8+ T cells response (p < 0.05). In conclusion, pH1N1 HA elicits a strong M-CD4+ T cells response in TMCs. Additionally, this response correlates with the response to sH1N1 suggesting cross-reactivity in T cells epitopes directed against HAs of both viral strains.

Characteristics of two zoonotic swine influenza A(H1N1) viruses isolated in Germany from diseased patients

Interspecies transmission of influenza A viruses (IAV) from pigs to humans is a concerning event as porcine IAV represent a reservoir of potentially pandemic IAV. We conducted a comprehensive analysis of two porcine A(H1N1)v viruses isolated from human cases by evaluating their genetic, antigenic and virological characteristics. The HA genes of those human isolates belonged to clades 1C.2.1 and 1C.2.2, respectively, of the A(H1N1) Eurasian avian-like swine influenza lineage. Antigenic profiling revealed substantial cross-reactivity between the two zoonotic H1N1 viruses and human A(H1N1)pdm09 virus and some swine viruses, but did not reveal cross-reactivity to H1N2 and earlier human seasonal A(H1N1) viruses. The solid-phase direct receptor binding assay analysis of both A(H1N1)v showed a predominant binding to α2–6–sialylated glycans similar to human-adapted IAV. Investigation of the replicative potential revealed that both A(H1N1)v viruses grow in human bronchial epithelial cells to similar high titers as the human A(H1N1)pdm09 virus. Cytokine induction was studied in human alveolar epithelial cells A549 and showed that both swine viruses isolated from human cases induced higher amounts of type I and type III IFN, as well as IL6 compared to a seasonal A(H1N1) or a A(H1N1)pdm09 virus. In summary, we demonstrate a remarkable adaptation of both zoonotic viruses to propagate in human cells. Our data emphasize the needs for continuous monitoring of people and regions at increased risk of such trans-species transmissions, as well as systematic studies to quantify the frequency of these events and to identify viral molecular determinants enhancing the zoonotic potential of porcine IAV.

Serological Monitoring of Pandemic Influenza Virus Markers in the Russian Federation in 2021–2023

State Scientific Center of Virology and Biotechnology “Vector” has been monitoring highly pathogenic influenza since 2005.The aim of this work was to track the markers of highly pathogenic influenza in the blood sera of people who had a contact with infected and/or deceased birds, as well as of residents from regions where emergence of new variants of influenza A virus is most likely to occur.Materials and methods. Sera were studied using hemagglutination inhibition test (HI test). HI-positive sera were subjected to virus neutralization reaction.Results and discussion. In 2021, 2076 blood serum samples from 19 regions of Russia were collected. Only 7 samples demonstrated significant titers in HI test with A/H5N8 viruses. In 2022, 1620 blood serum samples from 23 regions were obtained; 25 of them were positive for influenza А/H5N8 and А/H5N1 viruses. In 2023 (January-August), 3335 serum samples from 31 regions of the Russian Federation were collected. 28 samples were positive for influenza А/H5N8 and А/H5N1 viruses. Furthermore, we monitored blood sera for low-pathogenic A/H9N2 virus. The number of positive samples in 2021 was lower than 1 % (13 out of 2076); in 2022, it reached 5 % (81 out of 1620); in 2023, the share was lower than 1 % (31 out of 3335). The data obtained suggest indirectly that currently there is no stable circulation of zoonotic influenza A/H5N8 and A/H5N1 viruses in Russia. Influenza viruses A/H9N2 have widely spread in many countries of the world and actively participate in evolution of highly pathogenic influenza A/H5Nx viruses. The Russian Federation demonstrates a gradual increase in the number of blood serum samples with antibodies to A/H9N2 virus.

The influenza viruses in China: Pandemic and prevention measures

Seasonal influenza (SI) is a kind of respiratory virus with annual epidemic and high contagion, leading to cause increased morbidity and mortality in certain population. According to estimates, the flu causes an average of 400,000 respiratory deaths per year worldwide. The estimate mortality of flu is more than 65 per 100,000 people in the worldwide, and the ratio is 52.9/100,000 in China, demonstrating that seasonal influenza has caused a considerable disease burden in China. Influenza viruses are spread by droplets, and people infected with seasonal influenza usually have a sudden high fever, dry cough, and severe headache. Noteworthy, children and gravidas have higher rates of infection, and the elder population over 65 ages has a higher risks for complications, hospitalizations and death, especially patients with cardiovascular and pulmonary diseases. Therefore, prevention and treatment of high-risk populations is important to prevent and control influenza, while vaccination is an effective solution to protect them from infection and associated complications. In this paper, the author analyzes the epidemic features of influenza viruses in different regions of China in light of the prevention and control policies in various places, and provides some suggestions to prevent pandemic of influenza virus and enhance individuals preventive awareness, which purpose is to reduce the influenza positivity rate and the infection rate in the future, especially for vulnerable population of children and elder people.

Molecular modeling and phylogenetic analyses highlight the role of amino acid 347 of the N1 subtype neuraminidase in influenza virus host range and interspecies adaptation.

The N1 neuraminidases (NAs) of avian and pandemic human influenza viruses contain tyrosine and asparagine, respectively, at position 347 on the rim of the catalytic site; the biological significance of this difference is not clear. Here, we used molecular dynamics simulation to model the effects of amino acid 347 on N1 NA interactions with sialyllacto-N-tetraoses 6'SLN-LC and 3'SLN-LC, which represent NA substrates in humans and birds, respectively. Our analysis predicted that Y347 plays an important role in the NA preference for the avian-type substrates. The Y347N substitution facilitates hydrolysis of human-type substrates by resolving steric conflicts of the Neu5Ac2-6Gal moiety with the bulky side chain of Y347, decreasing the free energy of substrate binding, and increasing the solvation of the Neu5Ac2-6Gal bond. Y347 was conserved in all N1 NA sequences of avian influenza viruses in the GISAID EpiFlu database with two exceptions. First, the Y347F substitution was present in the NA of a specific H6N1 poultry virus lineage and was associated with the substitutions G228S and/or E190V/L in the receptor-binding site (RBS) of the hemagglutinin (HA). Second, the highly pathogenic avian H5N1 viruses of the Gs/Gd lineage contained sporadic variants with the NA substitutions Y347H/D, which were frequently associated with substitutions in the HA RBS. The Y347N substitution occurred following the introductions of avian precursors into humans and pigs with N/D347 conserved during virus circulation in these hosts. Comparative evolutionary analysis of site 347 revealed episodic positive selection across the entire tree and negative selection within most host-specific groups of viruses, suggesting that substitutions at NA position 347 occurred during host switches and remained under pervasive purifying selection thereafter. Our results elucidate the role of amino acid 347 in NA recognition of sialoglycan substrates and emphasize the significance of substitutions at position 347 as a marker of host range and adaptive evolution of influenza viruses.

Dihydrobenzoimidazothiazoles as novel anti-infective agents: Synthesis, biological evaluation and docking studies

Finding safer, more powerful and more effective molecules is urgently needed given the rising threat of infectious diseases. Benzoimidazothiazole are a novel class of small molecules that have not been studied on a variety of pharmacological targets. In this report, we discussed novel dihydrobenzo-imidazothiazole scaffold as promising anti-infective agents. Commercially available 1,3-cyclohexane-diones (1a–h) were brominated before being condensed with thiourea to produce 2-amino-5,6-dihydrobenzo[d]thiazol-7(4H)-ones (3a–h) in excellent yields. Compounds 3a-h were cyclized with 2‑bromo-1-(5-chlorothiophen-2-yl)ethan-1-one (4) to produce 2-(5-chlorothiophen-2-yl)-6,7-dihydrobenzo[d]Imidazo [2,1-b]thiazol-8(5H)-ones (5a-h). When synthetic analogues 3a-h and 5a-h were examined for biological activity, they showed effective inhibition against the tested antiviral and antibacterial strains. Two analogues, 3h (CC50: >300 µM, IC50= 8.3 µM, SI = >36) and 5h (CC50: >300 µM, IC50= 4.1 µM, SI = >73) had high levels of virus-inhibiting action when tested against the pandemic influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells with favorable toxicity profile. Among the substances tested against bacterial strains, 3d inhibited Typhi, Subtills, and Mutans bacteria with MICs of 0.78 µM and E. coli with MICs of 1.56 µM against standard streptomycin (MIC:1.56 µM). The novel analogues' physiochemical and molecular docking tests revealed promising pharmacological profiles for further structural optimization and drug development.

A novel “prime and pull” strategy mediated by the combination of two dendritic cell-targeting designs induced protective lung tissue-resident memory T cells against H1N1 influenza virus challenge

Vaccination is still the most promising strategy for combating influenza virus pandemics. However, the highly variable characteristics of influenza virus make it difficult to develop antibody-based universal vaccines, until now. Lung tissue-resident memory T cells (TRM), which actively survey tissues for signs of infection and react rapidly to eliminate infected cells without the need for a systemic immune reaction, have recently drawn increasing attention towards the development of a universal influenza vaccine. We previously designed a sequential immunization strategy based on orally administered Salmonella vectored vaccine candidates. To further improve our vaccine design, in this study, we used two different dendritic cell (DC)-targeting strategies, including a single chain variable fragment (scFv) targeting the surface marker DC-CD11c and DC targeting peptide 3 (DCpep3). Oral immunization with Salmonella harboring plasmid pYL230 (S230), which displayed scFv-CD11c on the bacterial surface, induced dramatic production of spleen effector memory T cells (TEM). On the other hand, intranasal boost immunization using purified DCpep3-decorated 3M2e-ferritin nanoparticles in mice orally immunized twice with S230 (S230inDC) significantly stimulated the differentiation of lung CD11b+ DCs, increased intracellular IL-17 production in lung CD4+ T cells and elevated chemokine production in lung sections, such as CXCL13 and CXCL15, as determined by RNAseq and qRT‒PCR assays, resulting in significantly increased percentages of lung TRMs, which could provide efficient protection against influenza virus challenge. The dual DC targeting strategy, together with the sequential immunization approach described in this study, provides us with a novel “prime and pull” strategy for addressing the production of protective TRM cells in vaccine design.

Development of Self-Assembled Protein Nanocage Spatially Functionalized with HA Stalk as a Broadly Cross-Reactive Influenza Vaccine Platform.

There remains a need for the development of a universal influenza vaccine, as current seasonal influenza vaccines exhibit limited protection against mismatched, mutated, or pandemic influenza viruses. A desirable approach to developing an effective universal influenza vaccine is the incorporation of highly conserved antigens in a multivalent scaffold that enhances their immunogenicity. Here, we develop a broadly cross-reactive influenza vaccine by functionalizing self-assembled protein nanocages (SAPNs) with multiple copies of the hemagglutinin stalk on the outer surface and matrix protein 2 ectodomain on the inner surface. SAPNs were generated by engineering short coiled coils, and the design was simulated by MD GROMACS. Due to the short sequences, off-target immune responses against empty SAPN scaffolds were not seen in immunized mice. Vaccination with the multivalent SAPNs induces high levels of broadly cross-reactive antibodies of only external antigens, demonstrating tight spatial control over the designed antigen placement. This work demonstrates the use of SAPNs as a potential influenza vaccine.