Sequential Immunization with Vaccines Based on SARS-CoV-2 Virus-like Particles Induces Broadly Neutralizing Antibodies.

Although many people have been vaccinated against COVID-19, infections with SARS-CoV-2 seem hard to avoid. There is a need to develop more effective vaccines and immunization strategies against emerging variants of infectious diseases. To understand whether different immunization strategies using variants sequence-based virus-like particles (VLPs) vaccines could offer superior immunity against future SARS-CoV-2 variants, our team constructed VLPs for the original Wuhan-Hu-1 strain (prototype), Delta (δ) variant, and Omicron (ο) variant of SARS-CoV-2, using baculovirus-insect expression system. Then we used these VLPs to assess the immune responses induced by homologous prime-boost, heterologous prime-boost, and sequential immunizations strategies in a mouse model. Our results showed that the pro+δ+ο sequential strategies elicited better neutralizing antibody responses. These sequential strategies also take advantage of inducing CD4+ T and CD8+ T lymphocytes proliferation and tendency to cytokine of Th1. Currently, our data suggest that sequential immunization with VLPs of encoding spike protein derived from SARS-CoV-2 variants of concern may be a potential vaccine strategy against emerging diseases, such as "Disease X".

A branching stochastic evolutionary model of the B-cell repertoire.

We propose a stochastic framework to describe the evolution of the B-cell repertoire during germinal center (GC) reactions. Our model is formulated as a multitype age-dependent branching process with time-varying immigration. The immigration process captures the mechanism by which founder B cells initiate clones by gradually seeding GC over time, while the branching process describes the temporal evolution of the composition of these clones. The model assigns a type to each cell to represent attributes of interest. Examples of attributes include the binding affinity class of the B cells, their clonal family, or the nucleotide sequence of the heavy and light chains of their receptors. The process is generally non-Markovian. We present its properties, including as t → ∞ when the process is supercritical, the most relevant case to study expansion of GC B cells. We introduce temporal alpha and beta diversity indices for multitype branching processes. We focus on the dynamics of clonal dominance, highlighting its non-stationarity, and the accumulation of somatic hypermutations in the context of sequential immunization. We evaluate the impact of the ongoing seeding of GC by founder B cells on the dynamics of the B-cell repertoire, and quantify the effect of precursor frequency and antigen availability on the timing of GC entry. An application of the model illustrates how it may help with interpretation of BCR sequencing data.

Immunogenicity evaluation of primary polio vaccination schedule with inactivated poliovirus vaccines and bivalent oral poliovirus vaccine.

BACKGROUND: To assess the immunogenicity of the current primary polio vaccination schedule in China and compare it with alternative schedules using Sabin or Salk-strain IPV (sIPV, wIPV). METHODS: A cross-sectional investigation was conducted at four sites in Chongqing, China, healthy infants aged 60-89 days were conveniently recruited and divided into four groups according to their received primary polio vaccination schedules (2sIPV + bOPV, 2wIPV + bOPV, 3sIPV, and 3wIPV). The sero-protection and neutralizing antibody titers against poliovirus serotypes (type 1, 2, and 3) were compared after the last dose. RESULTS: There were 408 infants completed the protocol. The observed seropositivity was more than 96% against poliovirus types 1, 2, and 3 in all groups. IPV-only groups induced higher antibody titers(GMT) against poliovirus type 2 (Median:192, QR: 96-384, P<0.05) than the "2IPV + bOPV" group. While the "2IPV + bOPV" group induced significantly higher antibody titers against poliovirus type 1 (Median:2048, QR: 768-2048, P<0.05)and type 3 (Median:2048, QR: 512-2048, P<0.05) than the IPV-only group. CONCLUSIONS: Our findings have proved that the two doses of IPV with one dose of bOPV is currently the best polio routine immunization schedule in China.

A Cocktail of Lipid Nanoparticle-mRNA Vaccines Broaden Immune Responses against ß-Coronaviruses in a Murine Model.

Severe acute respiratory syndrome (SARS)-coronavirus (CoV), Middle Eastern respiratory syndrome (MERS)-CoV, and SARS-CoV-2 have seriously threatened human life in the 21st century. Emerging and re-emerging ß-coronaviruses after the coronavirus disease 2019 (COVID-19) epidemic remain possible highly pathogenic agents that can endanger human health. Thus, pan-ß-coronavirus vaccine strategies to combat the upcoming dangers are urgently needed. In this study, four LNP-mRNA vaccines, named O, D, S, and M, targeting the spike protein of SARS-CoV-2 Omicron, Delta, SARS-CoV, and MERS-CoV, respectively, were synthesized and characterized for purity and integrity. All four LNP-mRNAs induced effective cellular and humoral immune responses against the corresponding spike protein antigens in mice. Furthermore, LNP-mRNA S and D induced neutralizing antibodies against SARS-CoV and SARS-CoV-2, which failed to cross-react with MERS-CoV. Subsequent evaluation of sequential and cocktail immunizations with LNP-mRNA O, D, S, and M effectively elicited broad immunity against SARS-CoV-2 variants, SARS-CoV, and MERS-CoV. A direct comparison of the sequential with cocktail regimens indicated that the cocktail vaccination strategy induced more potent neutralizing antibodies and T-cell responses against heterotypic viruses as well as broader antibody activity against pan-ß-coronaviruses. Overall, these results present a potential pan-ß-coronavirus vaccine strategy for improved preparedness prior to future coronavirus threats.

Heterologous Booster Immunization Based on Inactivated SARS-CoV-2 Vaccine Enhances Humoral Immunity and Promotes BCR Repertoire Development.

Recent studies have indicated that sequentially administering SARS-CoV-2 vaccines can result in increased antibody and cellular immune responses. In this study, we compared homologous and heterologous immunization strategies following two doses of inactivated vaccines in a mouse model. Our research demonstrates that heterologous sequential immunization resulted in more immune responses displayed in the lymph node germinal center, which induced a greater number of antibody-secreting cells (ASCs), resulting in enhanced humoral and cellular immune responses and increased cross-protection against five variant strains. In further single B-cell analysis, the above findings were supported by the presence of unique B-cell receptor (BCR) repertoires and diversity in CDR3 sequence profiles elicited by a heterologous booster immunization strategy.

Sequential Immunizations with Influenza Neuraminidase Protein Followed by Peptide Nanoclusters Induce Heterologous Protection.

Enhancing cross-protections against diverse influenza viruses is desired for influenza vaccinations. Neuraminidase (NA)-specific antibody responses have been found to independently correlate with a broader influenza protection spectrum. Here, we report a sequential immunization regimen that includes priming with NA protein followed by boosting with peptide nanoclusters, with which targeted enhancement of antibody responses in BALB/c mice to certain cross-protective B-cell epitopes of NA was achieved. The nanoclusters were fabricated via desolvation with absolute ethanol and were only composed of composite peptides. Unlike KLH conjugates, peptide nanoclusters would not induce influenza-unrelated immunity. We found that the incorporation of a hemagglutinin peptide of H2-d class II restriction into the composite peptides could be beneficial in enhancing the NA peptide-specific antibody response. Of note, boosters with N2 peptide nanoclusters induced stronger serum cross-reactivities to heterologous N2 and even heterosubtypic N7 and N9 than triple immunizations with the prototype recombinant tetrameric (rt) N2. The mouse challenge experiments with HK68 H3N2 also demonstrated the strong effectiveness of the peptide nanocluster boosters in conferring heterologous protection.

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.

Sequential heterologous immunization with COVID-19 vaccines induces broader neutralizing responses against SARS-CoV-2 variants in comparison with homologous boosters.

The recently prevalent variants of concerns (VOCs) of SARS-CoV-2 belong to Omicron variants which display increased transmissibility and evade from immune protection generated by vaccines and/or natural infections. Better immunization strategies should be explored to induce broader immune responses against evolving SARS-CoV-2 variants. Here, we used inactivated vaccines derived from ancestral (Wu), Delta (Del) and Omicron (Omi) strains to immunize mice with homologous booster (3 × Wu, 3 × Del and 3 × Omi) or heterologous sequential booster (Wu/Del/Omi and Omi/Wu/Del) to evaluate their responses against two pre-Omicron (Wu and Del) and four Omicron variants. Even though neutralization responses against Wu and Del variants were similar in heterologous and homologous immunization groups, heterologous immunization groups induced significantly stronger neutralizing antibody against BA.1 (4.1-11 folds higher) and BA.2 (4.7-14.2 folds higher) than those of homologous immunization groups. While homologous immunization only induced strong neutralizing responses to either pre-Omicron variants (Wu and Del) in 3 × Wu and 3 × Del groups or to Omicron variants (BA.1 and BA.2) in 3 × Omi group, heterologous immunization groups induced strong and broader neutralizing responses to both pre-Omicron (Wu, Del) and Omicron variants (BA.1 and BA.2). Homologous and heterologous immunization groups elicited similar antigen-specific T cell (IFN-γ+) and B cell responses. Compared with homologous immunization, heterologous immunization could induce stronger plasma cell responses, which have the potential to generate broader and stronger neutralizing antibodies. However, neither heterologous nor homologous immunization groups induced strong neutralizing antibody against variants with bigger genetic deviation, such as BA.4/5 or BF.7, only weak neutralizing responses were induced. Surveillance on SARS-CoV2 variants evolution and immunization strategy are needed to explore better vaccines with broader and stronger neutralizing antibodies against post pandemic COVID-19.

Chicken dendritic cell-targeting nanobodies mediated improved protective effects against H9N2 influenza virus challenge in a homologous sequential immunization study.

Global poultry production is still severely affected by H9N2 avian influenza virus (AIV), and the development of a novel universal AIV vaccine is still urgently needed. Neuraminidase (NA) has recently been shown to be an efficient conserved protective antigen. In this study, we fused the extracellular region of the NA gene with a ferritin cassette (pYL281), which resulted in self-assembled 24-mer nanoparticles with the NA protein displayed outside the nanoparticles. In addition, a chicken dendritic cell-targeting nanobody-phage74 was also inserted ahead of the NA protein to yield pYL294. Incubation with chicken bone marrow-derived dendritic cells (chBMDCs) showed that the DC-targeting nanoparticles purified from the pYL294 strain significantly increased the maturation of chBMDCs, as shown by increased levels of CCL5, CCR7, CD83 and CD86 compared with nontargeting proteins. Then, a chicken study was performed using Salmonella oral administration together with intranasal boost with purified proteins. Compared with the other groups, oral immunization with Salmonella harboring pYL294 followed by intranasal boost with purified DC-targeting nanoparticles dramatically increased the humoral IgY and mucosal IgA antibody response, as well as increased the cellular immune response, as shown by elevated splenic lymphocyte proliferation and intracellular mRNA levels of IL-4 and IFN-γ. Finally, sequential immunization with DC-targeting nanoparticles showed increased protection against G57 subtype H9N2 virus challenge compared with other groups, as shown by significantly decreased virus RNA copy numbers in oropharyngeal washes (Days 3, 5 and 7 post challenge) and cloacal washes (Day 7), significantly decreased lung virus titers on Day 5 post challenge and increased body weight gains during the challenge.

Enhancement of Neutralization Responses through Sequential Immunization of Stable Env Trimers Based on Consensus Sequences from Select Time Points by Mimicking Natural Infection.

HIV-1 vaccines have been challenging to develop, partly due to the high level of genetic variation in its genome. Thus, a vaccine that can induce cross-reactive neutralization activities will be needed. Studies on the co-evolution of antibodies and viruses indicate that mimicking the natural infection is likely to induce broadly neutralizing antibodies (bnAbs). We generated the consensus Env sequence for each time point in subject CH505, who developed broad neutralization activities, and selected five critical time points before broad neutralization was detected. These consensus sequences were designed to express stable Env trimers. Priming with the transmitted/founder Env timer and sequential boosting with these consensus Env trimers from different time points induced broader and more potent neutralizing activities than the BG505 Env trimer in guinea pigs. Analysis of the neutralization profiles showed that sequential immunization of Env trimers favored nAbs with gp120/gp41 interface specificity while the BG505 Env trimer favored nAbs with V2 specificity. The unique features such as consensus sequences, stable Env trimers and the sequential immunization to mimic natural infection likely has allowed the induction of improved neutralization responses.

Boost immunizations with NA-derived peptide conjugates achieve induction of NA inhibition antibodies and heterologous influenza protections.

Neuraminidase is suggested as an important component for developing a universal influenza vaccine. Targeted induction of neuraminidase-specific broadly protective antibodies by vaccinations is challenging. To overcome this, we rationally select the highly conserved peptides from the consensus amino acid sequence of the globular head domains of neuraminidase. Inspired by the B cell receptor evolution process, a reliable sequential immunization regimen is designed to result in immuno-focusing by steering bulk immune responses to a selected region where broadly protective B lymphocyte epitopes reside. After priming neuraminidase protein-specific antibody responses in C57BL/6 or BALB/c inbred mice strains by immunization or pre-infection, boost immunizations with certain neuraminidase-derived peptide-keyhole limpet hemocyanin conjugates significantly strengthened serum neuraminidase inhibition activities and cross-protections. Overall, this study provides proof of concept for a peptide-based sequential immunization strategy for achieving targeted induction of cross-protective antibody response, which provides references for designing universal vaccines against other highly variable pathogens.

Homologous Sequential Immunization Using Salmonella Oral Administration Followed by an Intranasal Boost with Ferritin-Based Nanoparticles Enhanced the Humoral Immune Response against H1N1 Influenza Virus.

The influenza virus continues to pose a great threat to public health due to the frequent variations in RNA viruses. Vaccines targeting conserved epitopes, such as the extracellular domain of the transmembrane protein M2 (M2e), a nucleoprotein, and the stem region of hemagglutinin proteins, have been developed, but more efficient strategies, such as nanoparticle-based vaccines, are still urgently needed. However, the labor-intensive in vitro purification of nanoparticles is still necessary, which could hinder the application of nanoparticles in the veterinary field in the future. To overcome this limitation, we used regulated lysis Salmonella as an oral vector with which to deliver three copies of M2e (3M2e-H1N1)-ferritin nanoparticles in situ and evaluated the immune response. Then, sequential immunization using Salmonella-delivered nanoparticles followed by an intranasal boost with purified nanoparticles was performed to further improve the efficiency. Compared with 3M2e monomer administration, Salmonella-delivered in situ nanoparticles significantly increased the cellular immune response. Additionally, the results of sequential immunization showed that the intranasal boost with purified nanoparticles dramatically stimulated the activation of lung CD11b dendritic cells (DCs) and elevated the levels of effector memory T (TEM) cells in both spleen and lung tissues as well as those of CD4 and CD8 tissue-resident memory T (TRM) cells in the lungs. The increased production of mucosal IgG and IgA antibody titers was also observed, resulting in further improvements to protection against a virus challenge, compared with the pure oral immunization group. Salmonella-delivered in situ nanoparticles efficiently increased the cellular immune response, compared with the monomer, and sequential immunization further improved the systemic immune response, as shown by the activation of DCs, the production of TEM cells and TRM cells, and the mucosal immune response, thereby providing us with a novel strategy by which to apply nanoparticle-based vaccines in the future. IMPORTANCE Salmonella-delivered in situ nanoparticle platforms may provide novel nanoparticle vaccines for oral administration, which would be beneficial for veterinary applications. The combination of administering Salmonella-vectored, self-assembled nanoparticles and an intranasal boost with purified nanoparticles significantly increased the production of effector memory T cells and lung resident memory T cells, thereby providing partial protection against an influenza virus challenge. This novel strategy could open a novel avenue for the application of nanoparticle vaccines for veterinary purposes.

Construction and immunogenicity of an mRNA vaccine against chikungunya virus.

Chikungunya fever (CHIKF) has spread to more than 100 countries worldwide, with frequent outbreaks in Europe and the Americas in recent years. Despite the relatively low lethality of infection, patients can suffer from long-term sequelae. Until now, no available vaccines have been approved for use; however, increasing attention is being paid to the development of vaccines against chikungunya virus (CHIKV), and the World Health Organization has included vaccine development in the initial blueprint deliverables. Here, we developed an mRNA vaccine using the nucleotide sequence encoding structural proteins of CHIKV. And immunogenicity was evaluated by neutralization assay, Enzyme-linked immunospot assay and Intracellular cytokine staining. The results showed that the encoded proteins elicited high levels of neutralizing antibody titers and T cell-mediated cellular immune responses in mice. Moreover, compared with the wild-type vaccine, the codon-optimized vaccine elicited robust CD8+ T-cell responses and mild neutralizing antibody titers. In addition, higher levels of neutralizing antibody titers and T-cell immune responses were obtained using a homologous booster mRNA vaccine regimen of three different homologous or heterologous booster immunization strategies. Thus, this study provides assessment data to develop vaccine candidates and explore the effectiveness of the prime-boost approach.

Immunogenicity of novel DNA vaccines encoding receptor-binding domain (RBD) dimer-Fc fusing antigens derived from different SARS-CoV-2 variants of concern.

The continuously emerging of severe acute respiratory syndrome coronavirus-2 variants of concern (VOCs) led to a decline in effectiveness of the first-generation vaccines. Therefore, optimized vaccines and vaccination strategies, which show advantages in protecting against VOCs, are urgently needed. Here we constructed an optimized DNA vaccine plasmid containing built-in CpG adjuvant, and designed vaccine candidates encoding five forms of antigens derived from Wuhan-Hu-1. The results showed that plasmid with receptor binding domain (RBD) dimer-Fc fusing antigen (2RBD-Fc) induced the highest level of RBD-specific IgG and neutralizing antibodies in mice. Then 2dRBD-Fc and 2omRBD-Fc vaccines, respectively derived from delta and omicron VOCs, were constructed. The 2dRBD-Fc induced potent humoral and cellular immune responses, while the immunogenicity of 2omRBD-Fc was low. We also observed that sequential immunization with 2RBD-Fc, 2dRBD-Fc and 2omRBD-Fc effectively elicited neutralizing antibodies against each immunized strain, and RBD-specific T cell responses. To be noted, the Wuhan-Hu-1, delta and omicron neutralizing antibody titers induced by sequential immunization were comparable to that induced by repetitive immunization with 2RBD-Fc, 2dRBD-Fc or 2omRBD-Fc respectively. The results suggest that sequential immunization with DNA vaccines encoding potent antigens derived from different VOCs, may be a promising strategy to elicit immune responses against multiple variants.

A fourth dose of Omicron RBD vaccine enhances broad neutralization against SARS-CoV-2 variants including BA.1 and BA.2 in vaccinated mice.

The SARS-CoV-2 vaccines have been widely used to build an immunologic barrier in the population against the COVID-19 pandemic. However, a newly emerging Omicron variant, including BA.1, BA.1.1, BA.2, and BA.3 sublineages, largely escaped the neutralization of existing neutralizing antibodies (nAbs), even those elicited by three doses of vaccines. Here, we used the Omicron BA.1 RBD as a fourth dose of vaccine to induce potent Omicron-specific nAbs and evaluated the broadly neutralizing activities against SARS-CoV-2 variants. The BA.1-based vaccine was indeed prone to induce a strain-specific antibody response substantially cross-reactive with BA.2 sublineage, and yet triggered broad neutralization against SARS-CoV-2 variants when it was used in the sequential immunization with WT and other variant vaccines. These results demonstrated that the booster of Omicron RBD vaccine could be a rational strategy to enhance the broadly nAb response.

Sequential immunization with SARS-CoV-2 RBD vaccine induces potent and broad neutralization against variants in mice.

The current COVID-19 pandemic caused by constantly emerging SARS-CoV-2 variants still poses a threat to public health worldwide. Effective next-generation vaccines and optimized booster vaccination strategies are urgently needed. Here, we sequentially immunized mice with a SARS-CoV-2 wild-type inactivated vaccine and a heterologous mutant RBD vaccine, and then evaluated their neutralizing antibody responses against variants including Beta, Delta, Alpha, Iota, Kappa, and A.23.1. These data showed that a third booster dose of heterologous RBD vaccine especially after two doses of inactivated vaccines significantly enhanced the GMTs of nAbs against all SARS-CoV-2 variants we tested. In addition, the WT and variants all displayed good cross-immunogenicity and might be applied in the design of booster vaccines to induce broadly neutralizing antibodies.

Exploration of a Sequential Gp140-Gp145 Immunization Regimen with Heterologous Envs to Induce a Protective Cross-Reactive HIV Neutralizing Antibody Response In Non-human Primates.

Raising a heterologous tier 2 neutralizing antibody (nAb) response remains a daunting task for HIV vaccine development. In this study, we explored the utility of diverse HIV-1 envelope (Env) immunogens in a sequential immunization scheme as a solution to this task. This exploration stemmed from the rationale that gp145, a membrane-bound truncation form of HIV Env, may facilitate the focusing of induced antibody response on neutralizing epitopes when sequentially combined with the soluble gp140 form as immunogens in a prime-boost mode. We first showed that gp140 DNA prime-gp145 Tiantan vaccinia (TV) boost likely represents a general format for inducing potent nAb response in mice. However, when examined in rhesus macaque, this modality showed little effectiveness. To improve the efficacy, we extended the original modality by adding a strong protein boost, namely native-like SOSIP.664 trimer displayed on ferritin-based nanoparticle (NP), which was generated by a newly developed click approach. The resulting three-immunization regimen succeeded in eliciting tier-2 nAb response with substantial breadth when implemented in rhesus macaque over a short 8-week schedule. Importantly, the elicited nAb response was able to effectively contain viremia upon a heterologous SHIV challenge. Collectively, our studies highlighted that diversification of Env immunogens, in both types and formulations, under the framework of a sequential immunization scheme might open new opportunity toward HIV vaccine development.

Successive Immunization With Epitope-Decreasing Dengue Antigens Induced Conservative Anti-Dengue Immune Responses.

Repeated homologous antigen immunization has been hypothesized to hinder antibody diversification, whereas sequential immunization with heterologous immunogens can educate B cell differentiations towards conserved residues thereby facilitating the generation of cross-reactive immunity. In this study, we developed a sequential vaccination strategy that utilized epitope-decreasing antigens to reinforce the cross-reactivity of T and B cell immune responses against all four serotypes dengue virus. The epitope-decreasing immunization was implemented by sequentially inoculating mice with antigens of decreasing domain complexity that first immunized with DENV1 live-attenuated virus, following by the Envelope protein (Env), and then Env domain III (EDIII) subunit protein. When compared to mice immunized with DENV1 live-attenuated virus three times, epitope-decreasing immunization induced higher TNF-α CD8+ T cell immune response against consensus epitopes. Epitope-decreasing immunization also significantly improved neutralizing antibody response to heterologous serotypes. Moreover, this sequential approach promoted somatic hypermutations in the immunoglobulin gene of antigen-specific memory B cells in comparison to repeated immunization. This proof-of-concept work on epitope-decreasing sequential vaccination sheds light on how successively exposing the immune system to decreasing-epitope antigens can better induce cross-reactive antibodies.

Sequential Immunization with Universal Live Attenuated Influenza Vaccine Candidates Protects Ferrets against a High-Dose Heterologous Virus Challenge.

The development of universal influenza vaccines has been a priority for more than 20 years. We conducted a preclinical study in ferrets of two sets of live attenuated influenza vaccines (LAIVs) expressing chimeric hemagglutinin (cHA). These vaccines contained the HA stalk domain from H1N1pdm09 virus but had antigenically unrelated globular head domains from avian influenza viruses H5N1, H8N4 and H9N2. The viral nucleoproteins (NPs) in the two sets of universal LAIV candidates were from different sources: one LAIV set contained NP from A/Leningrad/17 master donor virus (MDV), while in the other set this gene was from wild-type (WT) H1N1pdm09 virus, in order to better match the CD8 T-cell epitopes of currently circulating influenza A viruses. To avoid any difference in protective effect of the various anti-neuraminidase (NA) antibodies, all LAIVs were engineered to contain the NA gene of Len/17 MDV. Naïve ferrets were sequentially immunized with three doses of (i) classical LAIVs containing non-chimeric HA and NP from MDV (LAIVs (NP-MDV)); (ii) cHA-based LAIVs containing NP from MDV (cHA LAIVs (NP-MDV)); and (iii) cHA-based LAIVs containing NP from H1N1pdm09 virus (cHA LAIVs (NP-WT)). All vaccination regimens were safe, producing no significant increase in body temperature or weight loss, in comparison with the placebo group. The two groups of cHA-based vaccines induced a broadly reactive HA stalk-directed antibody, while classical LAIVs did not. A high-dose challenge with H1N1pdm09 virus induced significant pathology in the control, non-immunized ferrets, including high virus titers in respiratory tissues, clinical signs of disease and histopathological changes in nasal turbinates and lung tissues. All three vaccination regimens protected animals from clinical manifestations of disease: immunized ferrets did not lose weight or show clinical symptoms, and their fever was significantly lower than in the control group. Further analysis of virological and pathological data revealed the following hierarchy in the cross-protective efficacy of the vaccines: cHA LAIVs (NP-WT) > cHA LAIVs (NP-MDV) > LAIVs (NP-MDV). This ferret study showed that prototype universal cHA-based LAIVs are highly promising candidates for further clinical development.

Humanized Immunoglobulin Mice: Models for HIV Vaccine Testing and Studying the Broadly Neutralizing Antibody Problem.

A vaccine that can effectively prevent HIV-1 transmission remains paramount to ending the HIV pandemic, but to do so, will likely need to induce broadly neutralizing antibody (bnAb) responses. A major technical hurdle toward achieving this goal has been a shortage of animal models with the ability to systematically pinpoint roadblocks to bnAb induction and to rank vaccine strategies based on their ability to stimulate bnAb development. Over the past 6 years, immunoglobulin (Ig) knock-in (KI) technology has been leveraged to express bnAbs in mice, an approach that has enabled elucidation of various B-cell tolerance mechanisms limiting bnAb production and evaluation of strategies to circumvent such processes. From these studies, in conjunction with the wealth of information recently obtained regarding the evolutionary pathways and paratopes/epitopes of multiple bnAbs, it has become clear that the very features of bnAbs desired for their function will be problematic to elicit by traditional vaccine paradigms, necessitating more iterative testing of new vaccine concepts. To meet this need, novel bnAb KI models have now been engineered to express either inferred prerearranged V(D)J exons (or unrearranged germline V, D, or J segments that can be assembled into functional rearranged V(D)J exons) encoding predecessors of mature bnAbs. One encouraging approach that has materialized from studies using such newer models is sequential administration of immunogens designed to bind progressively more mature bnAb predecessors. In this review, insights into the regulation and induction of bnAbs based on the use of KI models will be discussed, as will new Ig KI approaches for higher-throughput production and/or altering expression of bnAbs in vivo, so as to further enable vaccine-guided bnAb induction studies.