Adam K. Wheatley

Immunological Principles Guiding the Rational Design of Particles for Vaccine Delivery

Despite the immense public health successes of immunization over the past century, effective vaccines are still lacking for globally important pathogens such as human immunodeficiency virus, malaria, and tuberculosis. Exciting recent advances in immunology and biotechnology over the past few decades have facilitated a shift from empirical to rational vaccine design, opening possibilities for improved vaccines. Some of the most important advancements include (i) the purification of subunit antigens with high safety profiles, (ii) the identification of innate pattern recognition receptors (PRRs) and cognate agonists responsible for inducing immune responses, and (iii) developments in nano- and microparticle fabrication and characterization techniques. Advances in particle engineering now allow highly tunable physicochemical properties of particle-based vaccines, including composition, size, shape, surface characteristics, and degradability. Enhanced collaborative efforts between researchers in immunology and materials science are expected to rise to next-generation vaccines. This process will be significantly aided by a greater understanding of the immunological principles guiding vaccine antigenicity, immunogenicity, and efficacy. With specific emphasis on PRR-targeted adjuvants and particle physicochemical properties, this review aims to provide an overview of the current literature to guide and focus rational particle-based vaccine design efforts.

Vaccine-Induced Antibodies that Neutralize Group 1 and Group 2 Influenza A Viruses.

Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and-in half the subjects-multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies.

Flow Cytometry Reveals that H5N1 Vaccination Elicits Cross-Reactive Stem-Directed Antibodies from Multiple Ig Heavy-Chain Lineages

ABSTRACT An understanding of the antigen-specific B-cell response to the influenza virus hemagglutinin (HA) is critical to the development of universal influenza vaccines, but it has not been possible to examine these cells directly because HA binds to sialic acid (SA) on most cell types. Here, we use structure-based modification of HA to isolate HA-specific B cells by flow cytometry and characterize the features of HA stem antibodies (Abs) required for their development. Incorporation of a previously described mutation (Y98F) to the receptor binding site (RBS) causes HA to bind only those B cells that express HA-specific Abs, but it does not bind nonspecifically to B cells, and this mutation has no effect on the binding of broadly neutralizing Abs to the RBS. To test the specificity of the Y98F mutation, we first demonstrated that previously described HA nanoparticles mediate hemagglutination and then determined that the Y98F mutation eliminates this activity. Cloning of immunoglobulin genes from HA-specific B cells isolated from a single human subject demonstrates that vaccination with H5N1 influenza virus can elicit B cells expressing stem monoclonal Abs (MAbs). Although these MAbs originated mostly from the IGHV1-69 germ line, a reasonable proportion derived from other genes. Analysis of stem Abs provides insight into the maturation pathways of IGVH1-69-derived stem Abs. Furthermore, this analysis shows that multiple non-IGHV1-69 stem Abs with a similar neutralizing breadth develop after vaccination in humans, suggesting that the HA stem response can be elicited in individuals with non-stem-reactive IGHV1-69 alleles. IMPORTANCE Universal influenza vaccines would improve immune protection against infection and facilitate vaccine manufacturing and distribution. Flu vaccines stimulate B cells in the blood to produce antibodies that neutralize the virus. These antibodies target a protein on the surface of the virus called HA. Flu vaccines must be reformulated annually, because these antibodies are mostly specific to the viral strains used in the vaccine. But humans can produce broadly neutralizing antibodies. We sought to isolate B cells whose genes encode influenza virus antibodies from a patient vaccinated for avian influenza. To do so, we modified HA so it would bind only the desired cells. Sequencing the antibody genes of cells marked by this probe proved that the patient produced broadly neutralizing antibodies in response to the vaccine. Many sequences obtained had not been observed before. There are more ways to generate broadly neutralizing antibodies for influenza virus than previously thought.

Abnormal B cell memory subsets dominate HIV-specific responses in infected individuals

Recently, several neutralizing anti-HIV antibodies have been isolated from memory B cells of HIV-infected individuals. Despite extensive evidence of B cell dysfunction in HIV disease, little is known about the cells from which these rare HIV-specific antibodies originate. Accordingly, we used HIV envelope gp140 and CD4 or coreceptor (CoR) binding site (bs) mutant probes to evaluate HIV-specific responses in peripheral blood B cells of HIV-infected individuals at various stages of infection. In contrast to non-HIV responses, HIV-specific responses against gp140 were enriched within abnormal B cells, namely activated and exhausted memory subsets, which are largely absent in the blood of uninfected individuals. Responses against the CoRbs, which is a poorly neutralizing epitope, arose early, whereas those against the well-characterized neutralizing epitope CD4bs were delayed and infrequent. Enrichment of the HIV-specific response within resting memory B cells, the predominant subset in uninfected individuals, did occur in certain infected individuals who maintained low levels of plasma viremia and immune activation with or without antiretroviral therapy. The distribution of HIV-specific responses among memory B cell subsets was corroborated by transcriptional analyses. Taken together, our findings provide valuable insight into virus-specific B cell responses in HIV infection and demonstrate that memory B cell abnormalities may contribute to the ineffectiveness of the antibody response in infected individuals.

Induction of HIV-1 subtype B and AE-specific neutralizing antibodies in mice and macaques with DNA prime and recombinant gp140 protein boost regimens.

We developed highly expressing clade B and AE DNA and envelope protein (Env) vaccines for evaluation in mice and macaques as DNA prime/protein boost regimens. High levels of Env-specific antibodies were induced in mice, albeit with limited neutralizing activity in vitro. A combined clade B and AE regimen induced high titer Env-specific antibody in two pigtail macaques that neutralized several strains of HIV-1. However, upon mucosal challenge with SHIV(SF162P3) no protection from infection was observed. Although the vaccines tested provide a platform for inducing robust humoral immunity, further refinements to broaden coverage against divergent strains and induce mucosal immunity are needed.

H5N1 Vaccine–Elicited Memory B Cells Are Genetically Constrained by the IGHV Locus in the Recognition of a Neutralizing Epitope in the Hemagglutinin Stem

Because of significant viral diversity, vaccines that elicit durable and broad protection against influenza have been elusive. Recent research has focused on the potential of highly conserved regions of the viral hemagglutinin (HA) as targets for broadly neutralizing Ab responses. Abs that bind the highly conserved stem or stalk of HA can be elicited by vaccination in humans and animal models and neutralize diverse influenza strains. However, the frequency and phenotype of HA stem–specific B cells in vivo remain unclear. In this article, we characterize HA stem–specific B cell responses following H5N1 vaccination and describe the re-expansion of a pre-existing population of memory B cells specific for stem epitopes. This population uses primarily, but not exclusively, IGHV1-69–based Igs for HA recognition. However, within some subjects, allelic polymorphism at the ighv1-69 locus can limit IGHV1-69 immunodominance and may reduce circulating frequencies of stem-reactive B cells in vivo. The accurate definition of allelic selection, recombination requirements, and ontogeny of neutralizing Ab responses to influenza will aid rational influenza vaccine design.

Hyperimmune Bovine Colostrum as a Low-Cost, Large-Scale Source of Antibodies with Broad Neutralizing Activity for HIV-1 Envelope with Potential Use in Microbicides

ABSTRACT Bovine colostrum (first milk) contains very high concentrations of IgG, and on average 1 kg (500 g/liter) of IgG can be harvested from each immunized cow immediately after calving. We used a modified vaccination strategy together with established production systems from the dairy food industry for the large-scale manufacture of broadly neutralizing HIV-1 IgG. This approach provides a low-cost mucosal HIV preventive agent potentially suitable for a topical microbicide. Four cows were vaccinated pre- and/or postconception with recombinant HIV-1 gp140 envelope (Env) oligomers of clade B or A, B, and C. Colostrum and purified colostrum IgG were assessed for cross-clade binding and neutralization against a panel of 27 Env-pseudotyped reporter viruses. Vaccination elicited high anti-gp140 IgG titers in serum and colostrum with reciprocal endpoint titers of up to 1 × 105. While nonimmune colostrum showed some intrinsic neutralizing activity, colostrum from 2 cows receiving a longer-duration vaccination regimen demonstrated broad HIV-1-neutralizing activity. Colostrum-purified polyclonal IgG retained gp140 reactivity and neutralization activity and blocked the binding of the b12 monoclonal antibody to gp140, showing specificity for the CD4 binding site. Colostrum-derived anti-HIV antibodies offer a cost-effective option for preparing the substantial quantities of broadly neutralizing antibodies that would be needed in a low-cost topical combination HIV-1 microbicide.

Antibody Responses with Fc-Mediated Functions after Vaccination of HIV-Infected Subjects with Trivalent Influenza Vaccine

ABSTRACT This study seeks to assess the ability of seasonal trivalent inactivated influenza vaccine (TIV) to induce nonneutralizing antibodies (Abs) with Fc-mediated functions in HIV-uninfected and HIV-infected subjects. Functional influenza-specific Ab responses were studied in 30 HIV-negative and 27 HIV-positive subjects immunized against seasonal influenza. All 57 subjects received the 2015 TIV. Fc-mediated antihemagglutinin (anti-HA) Ab activity was measured in plasma before and 4 weeks after vaccination using Fc-receptor-binding assays, NK cell activation assays, and phagocytosis assays. At baseline, the HIV-positive group had detectable but reduced functional Ab responses to both vaccine and nonvaccine influenza antigens. TIV enhanced Fc-mediated Ab responses in both HIV-positive and HIV-negative groups. A larger rise was generally observed in the HIV-positive group, such that there was no difference in functional Ab responses between the two groups after vaccination. The 2015 TIV enhanced functional influenza-specific Ab responses in both HIV-negative and HIV-positive subjects to a range of influenza HA proteins. The increase in functional Ab responses in the HIV-positive group supports recommendations to immunize this at-risk group. IMPORTANCE Infection with HIV is associated with increasing disease severity following influenza infections, and annual influenza vaccinations are recommended for this target group. However, HIV-infected individuals respond relatively poorly to vaccination compared to healthy individuals, particularly if immunodeficient. There is therefore a need to increase our understanding of immunity to influenza in the context of underlying HIV infection. While antibodies can mediate direct virus neutralization, interactions with cellular Fc receptors may be important for anti-influenza immunity in vivo by facilitating antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent phagocytosis (ADP). The ability of seasonal influenza vaccines to induce antibody responses with potent Fc-mediated antiviral activity is currently unclear. Probing the ADCC and ADP responses to influenza vaccination has provided important new information in the quest to improve immunity to influenza.

Efficient transcription through an intron requires the binding of an Sm-type U1 snRNP with intact stem loop II to the splice donor

The mechanism behind the positive action of introns upon transcription and the biological significance of this positive feedback remains unclear. Functional ablation of splice sites within an HIV-derived env cDNA significantly reduced transcription that was rescued by a U1 snRNA modified to bind to the mutated splice donor (SD). Using this model we further characterized both the U1 and pre-mRNA structural requirements for transcriptional enhancement. U1 snRNA rescued as a mature Sm-type snRNP with an intact stem loop II. Position and sequence context for U1-binding is crucial because a promoter proximal intron placed upstream of the mutated SD failed to rescue transcription. Furthermore, U1-rescue was independent of promoter and exon sequence and is partially replaced by the transcription elongation activator Tat, pointing to an intron-localized block in transcriptional elongation. Thus, transcriptional coupling of U1 snRNA binding to the SD may licence the polymerase for transcription through the intron.

Preferential induction of cross-group influenza A hemagglutinin stem–specific memory B cells after H7N9 immunization in humans

Broadly protective antibodies are preferentially induced upon vaccination with a group 2 immunogen. Stemming the tide of influenza A universal flu vaccine would prevent the need for yearly flu shots, but successful development has been hampered by the diversity and adaptability of the influenza virus. Andrews et al. compared B cell responses to the relatively conserved stem region of the influenza cell surface molecule hemagglutinin (HA) in humans vaccinated with either group 2 H7N9 or group 1 H5N1. They found that the stem-targeted memory B cells after H7N9 vaccination recognized both group 1 and group 2 influenza subtypes, whereas H5N1 vaccination induced responses primarily to group 1 subtypes. These data suggest that a group 2 stem immunogen, administered in the proper conditions, would have a higher likelihood of eliciting cross-group protection. Antigenic drift and shift of influenza strains underscore the need for broadly protective influenza vaccines. One strategy is to design immunogens that elicit B cell responses against conserved epitopes on the hemagglutinin (HA) stem. To better understand the elicitation of HA stem–targeted B cells to group 1 and group 2 influenza subtypes, we compared the memory B cell response to group 2 H7N9 and group 1 H5N1 vaccines in humans. Upon H7N9 vaccination, almost half of the HA stem–specific response recognized the group 1 and group 2 subtypes, whereas the response to H5N1 was largely group 1–specific. Immunoglobulin repertoire analysis of HA-specific B cells indicated that the H7N9 and H5N1 vaccines induced genetically similar cross-group HA stem–binding B cells, albeit at a much higher frequency upon H7N9 vaccination. These data suggest that a group 2–based stem immunogen could prove more effective than a group 1 immunogen at eliciting broad cross-group protection in humans.