Vaccine Development Research Articles

Nucleic acid vaccine candidates encapsulated with mesoporous silica nanoparticles against MERS-CoV

ABSTRACT Middle East respiratory coronavirus (MERS-CoV) is a newly emergent, highly pathogenic coronavirus that is associated with 34% mortality rate. MERS-CoV remains listed as priority pathogen by the WHO. Since its discovery in 2012 and despite the efforts to develop coronaviruses vaccines to fight against SARS-CoV-2, there are currently no MERS-CoV vaccine that has been approved. Therefore, there is high demand to continue on the development of prophylactic vaccines against MERS-CoV. Current advancements in vaccine developments can be adapted for the development of improved MERS-CoV vaccines candidates. Nucleic acid-based vaccines, including pDNA and mRNA, are relatively new class of vaccine platforms. In this work, we developed pDNA and mRNA vaccine candidates expressing S.FL gene of MERS-CoV. Further, we synthesized a silane functionalized hierarchical aluminosilicate to encapsulate each vaccine candidates. We tested the nucleic acid vaccine candidates in mice and evaluated humoral antibodies response. Interestingly, we determined that the non-encapsulated, codon optimized S.FL pDNA vaccine candidate elicited the highest level of antibody responses against S.FL and S1 of MERS-CoV. Encapsulation of mRNA with nanoporous aluminosilicate increased the humoral antibody responses, whereas encapsulation of pDNA did not. These findings suggests that MERS-CoV S.FL pDNA vaccine candidate induced the highest level of humoral responses. This study will enhance further optimization of nanosilica as potential carrier for mRNA vaccines. In conclusion, this study suggests MERS-CoV pDNA vaccine candidate as a suitable vaccine platform for further pivotal preclinical testings.

Prophylactic vaccination inducing anti-Env antibodies can result in protection against HTLV-1 challenge in macaques

HTLV-1 infection occurs by cell-to-cell transmission and can induce fatal adult T-cell leukemia (ATL). Vaccine development is critical for the control of HTLV-1 transmission. However, determining whether vaccine-induced anti-Env antibodies can prevent cell-to-cell HTLV-1 transmission is challenging. Here, we examined the protective efficacy of a vaccine inducing anti-Env antibodies against HTLV-1 challenge in cynomolgus macaques. Eight of ten vaccinated macaques produced anti-HTLV-1 neutralizing antibodies (NAbs) and were protected from an intravenous challenge with 108 HTLV-1-producing cells. In contrast, the two vaccinated macaques without NAb induction and ten unvaccinated controls showed HTLV-1 infection with detectable proviral load after challenge. Five of the eight protected macaques were administered with an anti-CD8 monoclonal antibody, but proviruses remained undetectable and no increase in anti-HTLV-1 antibodies was observed even after CD8+ cell depletion in three of them. Analysis of Env-specific T cell responses did not suggest involvement of vaccine-induced Env-specific T cell responses in the protection. These results indicate that anti-Env antibody induction by vaccination can result in functionally sterile HTLV-1 protection, implying the rationale for strategies aimed at anti-Env antibody induction in prophylactic HTLV-1 vaccine development.

Role of cytokines in immunomodulation during malaria clearance.

Malaria remains a significant global health challenge, demanding a deeper understanding of host immune responses for effective clearance of the parasitic infection. Cytokines, as crucial mediators of the immune system, orchestrate a complex interplay during the various stages of malaria infection. Throughout the course of the disease, an intricate balance of pro-inflammatory and anti-inflammatory cytokines dictate the immune response's outcome, influencing parasitic clearance and disease severity. During the initial stages, interleukins such as interleukin-12 (IL-12), interferon-gamma (IFN-γ), and tumour necrosis factor-alpha (TNF-α) play pivotal roles in activating innate immune cells, initiating the anti-parasitic response. Simultaneously, regulatory cytokines like interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β) modulate this immune activation, preventing excessive inflammation and tissue damage. As the infection progresses, a delicate shift occurs, characterized by a transition to adaptive immunity, guided by cytokines like interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13), promoting antibody production and T-cell responses. Notably, the resolution of malaria infection crucially relies on a fine-tuned balance of cytokine networks. Dysregulation or imbalances in these mediators often result in immune hyperactivation, contributing to severe manifestations and prolonged infection. Understanding the multi-faceted roles of cytokines in malaria clearance offers promising avenues for therapeutic interventions. Targeting cytokine pathways to restore immune equilibrium or bolster protective responses could potentially enhance treatment strategies and vaccine development. In conclusion, the pivotal role of cytokines in immunomodulation during malaria clearance underscores their significance as potential targets for therapeutic interventions, offering promising prospects in the global fight against this infectious disease.

Safety, tolerability, viral kinetics, and immune correlates of protection in healthy, seropositive UK adults inoculated with SARS-CoV-2: a single-centre, open-label, phase 1 controlled human infection study.

A SARS-CoV-2 controlled human infection model (CHIM) has been successfully established in seronegative individuals using a dose of 1×101 50% tissue culture infectious dose (TCID50) pre-alpha SARS-CoV-2 virus. Given the increasing prevalence of seropositivity to SARS-CoV-2, a CHIM that could be used for vaccine development will need to induce infection in those with pre-existing immunity. Our aim was to find a dose of pre-alpha SARS-CoV-2 virus that induced infection in previously infected individuals. Healthy, UK volunteers aged 18-30 years, with proven (quantitative RT-PCR or lateral flow antigen test) previous SARS-CoV-2 infection (with or without vaccination) were inoculated intranasally in a stepwise dose escalation CHIM with either 1×101, 1×102, 1×10³, 1×104, or 1×105 TCID50 SARS-CoV-2/human/GBR/484861/2020, the same virus used in the seronegative CHIM. Post-inoculation, volunteers were quarantined in functionally negative pressure rooms (Oxford, UK) for 14 days and until 12-hourly combined oropharyngeal-nasal swabs were negative for viable virus by focus-forming assay. Outpatient follow-up continued for 12 months post-enrolment, with additional visits for those who developed community-acquired SARS-CoV-2 infection. The primary objective was to identify a safe, well tolerated dose that induced infection (defined as two consecutive SARS-CoV-2 positive PCRs starting 24 h after inoculation) in 50% of seropositive volunteers. This study is registered with ClinicalTrials.gov (NCT04864548); enrolment and follow-up to 12 months post-enrolment are complete. Recruitment commenced on May 6, 2021, with the last volunteer enrolled into the dose escalation cohort on Nov 24, 2022. 36 volunteers were enrolled, with four to eight volunteers inoculated in each dosing group from 1×101 to 1×105 TCID50 SARS-CoV-2. All volunteers have completed quarantine, with follow-up to 12 months complete. Despite dose escalation to 1×105 TCID50, we were unable to induce sustained infection in any volunteers. Five (14%) of 36volunteers were considered to have transient infection, based on the kinetic of their PCR-positive swabs. Transiently infected volunteers had significantly lower baseline mucosal and systemic SARS-CoV-2-specific antibody titres and significantly lower peripheral IFNγ responses against a CD8+ T-cell SARS-CoV-2 peptide pool than uninfected volunteers. 14 (39%) of 36 volunteers subsequently developed breakthrough infection with the omicron variant after discharge from quarantine. Most adverse events reported by volunteers in quarantine were mild, with fatigue (16 [44%]) and stuffy nose (16 [44%]) being the most common. There were no serious adverse events. Our study demonstrates potent protective immunity induced by homologous vaccination and homologous or heterologous previous SARS-CoV-2 infection. The community breakthrough infections seen with the omicron variant supports the use of newer variants to establish a model with sufficient rate of infection for use in vaccine and therapeutic development. Wellcome Trust and Department for Health and Social Care.

A perspective on the worst ever dengue outbreak 2023 in Bangladesh: What makes this old enemy so deadly, and how can we combat it?

AbstractBackground and AimsBangladesh has been going through outbreaks of dengue fever cases every year since 2000. Yet this year's (2023) episode of dengue fever has crossed every line concerning fatality. Symptoms of the fever range from high fever, headaches, and muscle aches to deadly dengue hemorrhagic fever (DHF). The present review aims to assess the current pathogenicity and associated risk factors of recent dengue outbreaks in Bangladesh.MethodsTo perform this review work, we extracted relevant information from published articles available in PubMed, Scopus, and Google Scholar. We used dengue virus, dengue fever, and dengue outbreaks as keywords while searching for information.ResultsThis Aedes mosquito‐transmitted viral fever is more common in Bangladesh because of the tropical nature and immense burden of populations, resulting in convenient conditions for the reproduction of the vector. The rapid genetic transformation of this RNA virus and the resistance of its vector against insecticides have intensified the situation. The number of hospitalized patients has increased, and the case fatality rate has risen to 0.47%. Inadequate mosquito control measures, plenty of vector breeding sites, and a lack of public awareness have worsened the situation. Routine spraying of effective insecticides in high‐risk zones, regular inspection of potential mosquito breeding sites, and public awareness campaigns are the keys to limiting the spread of this virus. Also, the availability of detection kits, improved hospital settings, and trained health professionals are mandatory to keep disease fatalities under control.ConclusionDengue fever is a preventable disease. The successful development of a competent vaccine is now a prime need for preventing any future upsurge of the disease. Also, we recommend public awareness, vector control activities, and global collaboration to prevent spread.

MHC-Fine: Fine-tuned AlphaFold for Precise MHC-Peptide Complex Prediction

The precise prediction of Major Histocompatibility Complex (MHC)-peptide complex structures is pivotal for understanding cellular immune responses and advancing vaccine design. In this study, we enhanced AlphaFold’s capabilities by fine-tuning it with a specialized dataset consisting of exclusively high-resolution Class I MHC-peptide crystal structures. This tailored approach aimed to address the generalist nature of AlphaFold’s original training, which, while broad-ranging, lacked the granularity necessary for the high-precision demands of Class I MHC-peptide interaction prediction. A comparative analysis was conducted against the homology-modeling-based method Pandora, as well as the AlphaFold multimer model. Our results demonstrate that our fine-tuned model outperforms others in terms of RMSD (median value for C-alpha atoms for peptides is 0.66 Å) and also provides enhanced predicted lDDT scores, offering a more reliable assessment of the predicted structures. These advances have substantial implications for computational immunology, potentially accelerating the development of novel therapeutics and vaccines by providing a more precise computational lens through which to view MHC-peptide interactions.

Virus-like particle-based multipathogen vaccine of FMD and SVA elicits balanced and broad protective efficacy in mice and pigs

Inactivated vaccines lack the capability to serologically differentiate between infected and vaccinated animals, thereby impeding the effective eradication of pathogen. Conversely, vaccines based on virus-like particles (VLPs) emulate natural viruses in both size and antigenic structure, presenting a promising alternative to overcome these limitations. As the complexity of swine infectious diseases increases, the increase of vaccine types and doses may intensify the stress response. This exacerbation can lead to diminished productivity, failure of immunization, and elevated costs. Given the critical dynamics of co-infection and the clinically indistinguishable symptoms associated with foot-and-mouth disease virus (FMDV) and senecavirus A (SVA), there is a dire need for an efficacious intervention. To address these challenges, we developed a combined vaccine composed of three distinct VLPs, specifically designed to target SVA and FMDV serotypes O and A. Our research demonstrates that this trivalent VLP vaccine induces antigen-specific and robust serum antibody responses, comparable to those produced by the respective monovalent vaccines. Moreover, the immune sera from the combined VLP vaccine strongly neutralized FMDV type A and O, and SVA, with neutralization titers comparable to those of the individual vaccines, indicating a high level of immunogenic compatibility among the three VLP components. Importantly, the combined VLPs vaccines-immunized sera conferred efficient protection against single or mixed infections with FMDV type A and O, and SVA viruses in pigs. In contrast, individual vaccines could only protect pigs against homologous virus infections and not against heterologous challenges. This study presents a novel combined vaccines candidate against FMD and SVA, and provides new insights for the development of combination vaccines for other viral swine diseases.

A Glance on Nanovaccine: A Potential Approach for Disease Prevention.

There are several vaccines available for preventing various bacterial and viral infections, but still, there are many challenges that require the development of noninvasive, more efficient, and active vaccines. The advancement in biotechnological tools has provided safer antigens, such as nucleic acids, proteins etc., but due to their lower immunogenic property, adjuvants of stronger immune response are required. Nanovaccines are effective vaccines when compared with conventional vaccines as they can induce both Humoral and cell-mediated immune responses and also provide longer immunogenic memory. The nanocarriers used in vaccines act as adjuvant. They provide site-specific delivery of antigens and can be used in conjugation with immunostimulatory molecules for enhancing adjuvant therapy. The nanovaccines avoid degrading cell pathways and provide effective absorption into blood vessels. The higher potential of nanovaccines to treat various diseases, such as Acquired Immuno Deficiency Syndrome, Cancer, Tuberculosis, Malaria and many others, along with their immunological mechanisms and different types, have been discussed in the review.

Campylobacter jejuni virulence factors: update on emerging issues and trends.

Campylobacter jejuni is a very common cause of gastroenteritis, and is frequently transmitted to humans through contaminated food products or water. Importantly, C. jejuni infections have a range of short- and long-term sequelae such as irritable bowel syndrome and Guillain Barre syndrome. C. jejuni triggers disease by employing a range of molecular strategies which enable it to colonise the gut, invade the epithelium, persist intracellularly and avoid detection by the host immune response. The objective of this review is to explore and summarise recent advances in the understanding of the C. jejuni molecular factors involved in colonisation, invasion of cells, collective quorum sensing-mediated behaviours and persistence. Understanding the mechanisms that underpin the pathogenicity of C. jejuni will enable future development of effective preventative approaches and vaccines against this pathogen.

Comprehensive observational study evaluating the enduring effectiveness of 4CMenB, the meningococcal B vaccine against gonococcal infections in the Northern Territory and South Australia, Australia: study protocol

IntroductionThe effectiveness of antibiotics for treating gonococcal infections is compromised due to escalating antibiotic resistance; and the development of an effective gonococcal vaccine has been challenging. Emerging evidence suggests that...

Development of Multi-epitope mRNA Vaccine against Clostridioides difficile Using Reverse Vaccinology and Immunoinformatics Approaches

Development of Multi-epitope mRNA Vaccine against Clostridioides difficile Using Reverse Vaccinology and Immunoinformatics Approaches

Plug-and-display nanoparticle immunization of the core epitope domain induces potent neutralizing antibody and cellular immune responses against PEDV

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, is widely spread worldwide and causes huge economic losses. The effective measure to control the viral infection is to develop ideal vaccines. Here, the monomeric core epitope domain (COE) of PEDV was displayed on the surface of nanoparticles (NPs) in order to develop a newer, safer and more effective subunit vaccine against PEDV. The monomeric COE was displayed on the mi3 protein, which self-assembles into nanoparticles composed of 60 subunits, using the SpyTag/SpyCatcher system. The size, Zeta potential, microstructure of the COE-mi3 virus-like particles (VLPs)were investigated. The COE-mi3 VLPs that possessed good security, stability and better retention can be more efficiently taken up by antigen-presenting cells (APCs) and help promote dendritic cells (DCs) maturation. Moreover, COE-mi3 VLPs could prominently improve specific antibody levels including neutralizing antibodies (NAbs), and serum IgG, mucosal IgA. Moreover, COE-mi3 VLPs elicited more activation of CD4+ and CD8+ T cells and production of IFN-γ and IL-4 cytokines. In particular, COE-mi3 VLPs is an effectual antigen-delivery platform to enhance germinal center (GC) B cell responses. This structure-based self-assembly of NP gives great potential to be developed as a new subunit vaccines attractive platform, and may also provide new ideas for the development of other enteric coronavirus vaccines.

Identification of B-cell epitope on the N protein of type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using monoclonal antibody and construction of epitope-mutated virus

The escalating epidemic of PRRSV-1 in China has prompted widespread concern regarding the evolution of strains, disparities in pathogenicity to herds, and immunological detection of emerging strains. The nucleocapsid (N) protein, as a highly conserved protein with immunogenic properties in PRRSV, is a subject of intensive study. In this research, the recombinant His-N protein was expressed based on the N gene of PRRSV-1 using a prokaryotic expression system and then administered to BALB/c mice. A cell fusion protocol was implemented between SP2/0 cells and splenocytes, resulting in the successful screening of a monoclonal antibody against the N protein, designated as mAb 2D7, by indirect ELISA. Western Blot analysis and Indirect Immunofluorescence Assay (IFA) confirmed that mAb 2D7 positively responded to PRRSV-1. By constructing and expressing a series of truncated His-fused N proteins, a B-cell epitope of N protein, 59-AAEDDIR-65, was identified. A sequence alignment of two genotypes of PRRSV revealed that this epitope is relatively conserved in PRRSV, yet more so in genotype 1. Cross-reactivity analysis by Western blot analysis demonstrated that the B-cell epitope containing D62Y mutation could not be recognized by mAb 2D7. The inability of mAb 2D7 to recognize the epitope carrying the D62Y mutation was further determined using an infectious clone of PRRSV. This research may shed light on the biological significance of the N protein of PRRSV, paving the way for the advancement of immunological detection and development of future recombinant marker vaccine.

An mRNA technology transfer programme and economic sustainability in health care.

The World Health Organization (WHO) set up the messenger ribonucleic acid (mRNA) technology transfer programme in June 2021 with a development hub in South Africa and 15 partner vaccine producers in middle-income countries. The goal was to support the sustainable development of and access to life-saving vaccines for people in these countries as a means to enhance epidemic preparedness and global public health. This initiative aims to build resilience and strengthen local vaccine research, and development and manufacturing capacity in different regions of the world, especially those areas that could not access coronavirus disease 2019 (COVID-19) vaccines in a timely way. This paper outlines the current global vaccine market and summarizes the findings of a case study on the mRNA technology transfer programme conducted from November 2022 to May 2023. The study was guided by the vision of the WHO Council on the Economics of Health for All to build an economy for health using its four work streams of value, finance, innovation and capacity. Based on the findings of the study, we offer a mission-oriented policy framework to support the mRNA technology transfer programme as a pilot for transformative change towards an ecosystem for health innovation for the common good. Parts of this vision have already been incorporated into the governance of the mRNA technology transfer programme, while other aspects, especially the common good approach, still need to be applied to achieve the goals of the programme.

Targeting PfProhibitin 2-Hu-Hsp70A1A complex as a unique approach towards malaria vaccine development

Targeting PfProhibitin 2-Hu-Hsp70A1A complex as a unique approach towards malaria vaccine development

Vaccine Developers Leverage mRNA and Other Powerful Technologies

Vaccine Developers Leverage mRNA and Other Powerful Technologies

Guiding a language-model based protein design method towards MHC Class-I immune-visibility targets in vaccines and therapeutics

Proteins have an arsenal of medical applications that include disrupting protein interactions, acting as potent vaccines, and replacing genetically deficient proteins. While therapeutics must avoid triggering unwanted immune-responses, vaccines should support a robust immune-reaction targeting a broad range of pathogen variants. Therefore, computational methods modifying proteins’ immunogenicity without disrupting function are needed. While many components of the immune-system can be involved in a reaction, we focus on Cytotoxic T-lymphocytes (CTLs). These target short peptides presented via the MHC Class I (MHC-I) pathway. To explore the limits of modifying the visibility of those peptides to CTLs within the distribution of naturally occurring sequences, we developed a novel machine learning technique, CAPE-XVAE. It combines a language model with reinforcement learning to modify a protein’s immune-visibility. Our results show that CAPE-XVAE effectively modifies the visibility of the HIV Nef protein to CTLs. We contrast CAPE-XVAE to CAPE-Packer, a physics-based method we also developed. Compared to CAPE-Packer, the machine learning approach suggests sequences that draw upon local sequence similarities in the training set. This is beneficial for vaccine development, where the sequence should be representative of the real viral population. Additionally, the language model approach holds promise for preserving both known and unknown functional constraints, which is essential for the immune-modulation of therapeutic proteins. In contrast, CAPE-Packer, emphasizes preserving the protein’s overall fold and can reach greater extremes of immune-visibility, but falls short of capturing the sequence diversity of viral variants available to learn from. Source code: https://github.com/hcgasser/CAPE (Branch: CAPE_1.1)

Immunogenicity and immunodominant linear B-cell epitopes of a new DNA-based tetravalent vaccine against four major enteroviruses causing hand, foot, and mouth disease

Hand, foot, and mouth disease (HFMD) poses a significant public health threat primarily caused by four major enteroviruses: enterovirus 71 (EV71), coxsackieviruses A16, A10, and A6. Broadly protective immune responses are essential for complete protection against these major enteroviruses. In this study, we designed a new tetravalent immunogen for HFMD, validated it in silico, in vivo evaluated the immunogenicity of the DNA-based tetravalent vaccine in mice, and identified immunogenic B-cell and T-cell epitopes. A new tetravalent immunogen, VP1me, was designed based on the chimeric protein and epitope-based vaccine principles. It contains a complete EV71 VP1 protein and six reported neutralizing B-cell epitopes derived from the four major enteroviruses causing HFMD. In silico validation using multiple immunoinformatic tools indicated good attributes of the VP1me immunogen suitable for vaccine development. The VP1me-based DNA vaccine efficiently induced both humoral and cellular immune responses in BALB/cAJcl mice. A combination of in silico prediction and immunoassays enabled the identification of immunogenic linear B-cell and CD8 T-cell epitopes within the VP1me immunogen. Immunodominant linear B-cell epitopes were identified in six regions of VP1me, with one epitope located at the N-terminus of the VP1 protein (aa 9–23) regarded as a novel epitope. Interestingly, some B-cell epitopes could also induce the CD8 T-cell response, suggesting their dual functions in immune stimulation. These results lay the groundwork for further development of VP1me as a new vaccine candidate.

A 3D organoid platform that supports liver-stage P.falciparum infection can be used to identify intrahepatic antimalarial drugs

Malaria, a major public health burden, is caused by Plasmodium spp parasites that first replicate in the human liver to establish infection before spreading to erythrocytes. Liver-stage malaria research has remained challenging due to the lack of a clinically relevant and scalable in vitro model of the human liver. Here, we demonstrate that organoids derived from intrahepatic ductal cells differentiated into a hepatocyte-like fate can support the infection and intrahepatic maturation of Plasmodium falciparum. The P.falciparum exoerythrocytic forms observed expressed both early and late-stage parasitic proteins and decreased in frequency in response to treatment with both known and putative antimalarial drugs that target intrahepatic P.falciparum. The P.falciparum-infected human liver organoids thus provide a platform not only for fundamental studies that characterise intrahepatic parasite-host interaction but can also serve as a powerful translational tool in pre-erythrocytic vaccine development and to identify new antimalarial drugs that target the liver stage infection.

Effect of human H3N2 influenza virus reassortment on influenza incidence and severity during the 2017–18 influenza season in the USA: a retrospective observational genomic analysis

During the 2017-18 influenza season in the USA, there was a high incidence of influenza illness and mortality. However, no apparent antigenic change was identified in the dominant H3N2 viruses, and the severity of the season could not be solely attributed to a vaccine mismatch. We aimed to investigate whether the altered virus properties resulting from gene reassortment were underlying causes of the increased case number and disease severity associated with the 2017-18 influenza season. Samples included were collected from patients with influenza who were prospectively recruited during the 2016-17 and 2017-18 influenza seasons at the Johns Hopkins Hospital Emergency Departments in Baltimore, MD, USA, as well as from archived samples from Johns Hopkins Health System sites. Among 647 recruited patients with influenza A virus infection, 411 patients with whole-genome sequences were available in the Johns Hopkins Center of Excellence for Influenza Research and Surveillance network during the 2016-17 and 2017-18 seasons. Phylogenetic trees were constructed based on viral whole-genome sequences. Representative viral isolates of the two seasons were characterised in immortalised cell lines and human nasal epithelial cell cultures, and patients' demographic data and clinical outcomes were analysed. Unique H3N2 reassortment events were observed, resulting in two predominant strains in the 2017-18 season: HA clade 3C.2a2 and clade 3C.3a, which had novel gene segment constellations containing gene segments from HA clade 3C.2a1 viruses. The reassortant re3C.2a2 viruses replicated with faster kinetics and to a higher peak titre compared with the parental 3C.2a2 and 3C.2a1 viruses (48 h vs 72 h). Furthermore, patients infected with reassortant 3C.2a2 viruses had higher Influenza Severity Scores than patients infected with the parental 3C.2a2 viruses (median 3·00 [IQR 1·00-4·00] vs 1·50 [1·00-2·00]; p=0·018). Our findings suggest that the increased severity of the 2017-18 influenza season was due in part to twointrasubtypes, cocirculating H3N2 reassortant viruses with fitness advantages over the parental viruses. This information could help inform future vaccine development and public health policies. The Center of Excellence for Influenza Research and Response in the US, National Science and Technology Council, and Chang Gung Memorial Hospital in Taiwan.