Flora Castellino

MF59 Adjuvant Enhances Diversity and Affinity of Antibody-Mediated Immune Response to Pandemic Influenza Vaccines

Adjuvant use improves the quality and quantity of the immune response to pandemic influenza vaccines. A Shot in the Arm for the Flu Vaccine Because flu vaccine production begins months before the flu season, vaccine producers and world health officials have to guess which strains will predominate in a coming year. Although they’re frequently right, sometimes a dark horse arises that wasn’t predicted, and vaccine makers have to play catch-up. Speed is of the essence when attempting to prevent a pandemic, and limited supplies could hinder a worldwide vaccination campaign, preventing the broad coverage that protects even unvaccinated people. Khurana et al. report that the use of an oil-in-water adjuvant, MF59, improves the magnitude, robustness, and strength of the immune response to pandemic flu vaccines and may decrease the amount of antigen required for each dose. The authors looked at the quantitative and qualitative effects of MF59 on the antibody response in patients from various age groups who were vaccinated with the swine-origin H1N1 flu vaccine. They found that in adults and children who had been either previously vaccinated or exposed to the flu, MF59 increased the diversity and volume of neutralizing antibody responses to the flu vaccine. Antibody responses were slightly different in toddlers, suggesting that there may be distinct requirements for activating primary and recall antibody responses. MF59 also increased the strength with which these antibodies bound to the flu virus. These results support the use of adjuvants with flu vaccinations. Such a strategy could help expand coverage and decrease morbidity in the event of pandemic influenza and give vaccine makers the edge in a game of catch-up with dark horses. Oil-in-water adjuvants have been shown to improve immune responses against pandemic influenza vaccines as well as reduce the effective vaccine dose, increasing the number of doses available to meet global vaccine demand. Here, we use genome fragment phage display libraries and surface plasmon resonance to elucidate the effects of MF59 on the quantity, diversity, specificity, and affinity maturation of human antibody responses to the swine-origin H1N1 vaccine in different age groups. In adults and children, MF59 selectively enhanced antibody responses to the hemagglutinin 1 (HA1) globular head relative to the more conserved HA2 domain in terms of increased antibody titers as well as a more diverse antibody epitope repertoire. Antibody affinity, as inferred by greatly diminished (≥10-fold) off-rate constants, was significantly increased in toddlers and children who received the MF59-adjuvanted vaccine. Moreover, MF59 also improved antibody affinity maturation after each sequential vaccination against avian H5N1 in adults. For both pandemic influenza vaccines, there was a close correlation between serum antibody affinity and virus-neutralizing capacity. Thus, MF59 quantitatively and qualitatively enhances functional antibody responses to HA-based vaccines by improving both epitope breadth and binding affinity, demonstrating the added value of such adjuvants for influenza vaccines.

Chemokines enhance immunity by guiding naive CD8 + T cells to sites of CD4 + T cell–dendritic cell interaction

CD8+ T cells have a crucial role in resistance to pathogens and can kill malignant cells; however, some critical functions of these lymphocytes depend on helper activity provided by a distinct population of CD4+ T cells. Cooperation between these lymphocyte subsets involves recognition of antigens co-presented by the same dendritic cell, but the frequencies of such antigen-bearing cells early in an infection and of the relevant naive T cells are both low. This suggests that an active mechanism facilitates the necessary cell–cell associations. Here we demonstrate that after immunization but before antigen recognition, naive CD8+ T cells in immunogen-draining lymph nodes upregulate the chemokine receptor CCR5, permitting these cells to be attracted to sites of antigen-specific dendritic cell–CD4+ T cell interaction where the cognate chemokines CCL3 and CCL4 (also known as MIP-1α and MIP-1β) are produced. Interference with this actively guided recruitment markedly reduces the ability of CD4+ T cells to promote memory CD8+ T-cell generation, indicating that an orchestrated series of differentiation events drives nonrandom cell–cell interactions within lymph nodes, optimizing CD8+ T-cell immune responses involving the few antigen-specific precursors present in the naive repertoire.

Adjuvanted H5N1 vaccine induces early CD4+ T cell response that predicts long-term persistence of protective antibody levels

Immune responses to vaccination are tested in clinical trials. This process usually requires years especially when immune memory and persistence are analyzed. Markers able to quickly predict the immune response would be very useful, particularly when dealing with emerging diseases that require a rapid response, such as avian influenza. To address this question we vaccinated healthy adults at days 1, 22, and 202 with plain or MF59-adjuvanted H5N1 subunit vaccines and tested both cell-mediated and antibody responses up to day 382. Only the MF59-H5N1 vaccine induced high titers of neutralizing antibodies, a large pool of memory H5N1-specific B lymphocytes, and H5-CD4+ T cells broadly reactive with drifted H5. The CD4+ response was dominated by IL-2+ IFN-γ− IL-13− T cells. Remarkably, a 3-fold increase in the frequency of virus-specific total CD4+ T cells, measurable after 1 dose, accurately predicted the rise of neutralizing antibodies after booster immunization and their maintenance 6 months later. We suggest that CD4+ T cell priming might be used as an early predictor of the immunogenicity of prepandemic vaccines.

Fast rise of broadly cross-reactive antibodies after boosting long-lived human memory B cells primed by an MF59 adjuvanted prepandemic vaccine

Proactive priming before the next pandemic could induce immune memory responses to novel influenza antigens. In an open-label study, we analyzed B cell memory and antibody responses of 54 adults who received 2 7.5-μg doses of MF59-adjuvanted A/Vietnam/1194/2004 clade 1 (H5N1) vaccine. Twenty-four subjects had been previously primed with MF59-adjuvanted or plain clade 0-like A/duck/Singapore/1997 (H5N3) vaccine during 1999–2001. The prevaccination frequency of circulating memory B cells reactive to A/Vietnam/1194/2004 was low in both primed and unprimed individuals. However, at day 21 after boosting, MF59-adjuvanted primed subjects displayed a higher frequency of H5N1-specific memory B cells than plain-primed or unprimed subjects. The immune memory was rapidly mobilized by a single vaccine administration and resulted in high titers of neutralizing antibodies to antigenically diverse clade 0, 1, and 2 H5N1 viruses already at day 7. In general, postvaccination antibody titers were significantly higher in primed subjects than in unprimed subjects. Subjects primed with MF59-adjuvanted vaccine responded significantly better than those primed with plain vaccine, most notably in early induction and duration of cross-reacting antibody responses. After 6 months, high titers of cross-reactive antibody remained detectable among MF59-primed subjects. We conclude that distant priming with clade 0-like H5N3 induces a pool of cross-reactive memory B cells that can be boosted rapidly years afterward by a mismatched MF59-adjuvanted vaccine to generate high titers of cross-reactive neutralizing antibodies rapidly. These results suggest that pre-pandemic vaccination strategies should be considered.

Vaccines with MF59 Adjuvant Expand the Antibody Repertoire to Target Protective Sites of Pandemic Avian H5N1 Influenza Virus

An oil-based adjuvant improves the efficacy of an H5N1 vaccine by inducing antibodies against additional sites on influenza surface proteins. Revving Up a Flu Vaccine By preventing incalculable illnesses, vaccines are one of medicine’s great triumphs. Part of the credit goes to the adjuvants, substances included in vaccine preparations that boost the immune response but have no effect on their own. These agents—as various as aluminum salts and lipids—are thought to activate the innate immune system, the generalized protective response most animals show to pathogens. But the precise mechanisms by which these agents augment the immune response continue to elude scientists. Khurana and colleagues have now closely compared the immune response to an avian influenza vaccine without adjuvant to that induced with an oil-in-water adjuvant. Mixing the adjuvant with the vaccine induced antibodies that recognized a wider variety of flu antigens, including some known to inactivate the virus. The adjuvant examined by these authors was MF59—an emulsion of squalene, a natural hydrocarbon, and several fatty acids—which is used to boost influenza virus vaccines marketed in Europe. Flu vaccines that include MF59 engender more neutralizing antibodies than those that do not. To better understand the characteristics of these additional antibodies to flu proteins, the authors analyzed sera from subjects in two clinical trials of vaccines against the avian flu H5N1, with and without MF59. As expected, MF59 increased the amount of antibody against H5N1 flu, but the diversity of the new antibodies was also amplified. The authors looked specifically for antibodies directed at regions of the hemagglutinin protein and at neuraminidase, through which the virus binds to and infects its host’s cells. [The H5N1 name of the influenza strain refers to the exact type of hemagglutinin (H) and neuraminidase (N) proteins carried by the virus.] MF59 caused production of more antibodies directed against the hemagglutinin region that binds host cells, rather than the region that anchors the protein in the membrane, and many more regions within these areas were targeted. Only vaccines with MF59 generated antibodies against long sequences and against the native conformation of the host cell binding region. These particular antibodies are important because they are the ones that successfully block infection—the ultimate goal of any vaccine. They were also able to bind to and neutralize hemagglutinin from other strains of H5N1 flu—from Indonesia and China—much more effectively than were antibodies induced by vaccines without adjuvant; this finding showed that MF59-containing vaccines also have the desirable feature of extending protection beyond the particular flu strain used for vaccination. This study does not reveal exactly how the wider immune repertoire induced by MF59 is generated. But it does begin to define precisely the nature of the adjuvant-induced antibodies to influenza and to explain why they are more effective than those made in response to vaccines without adjuvant. MF59 is the adjuvant used in some vaccines against the current pandemic H1N1 flu, and the conclusions from this study are likely to apply to those vaccines as well. Vaccines against influenza viruses with pandemic potential, including H5N1, are under development. Because of a lack of preexisting immunity to these viruses, adjuvants (immune potentiators or enhancers) are needed to improve immune responses, to conserve scarce vaccine, and for cross-protection against strains that have drifted evolutionarily from the original. Aluminum-based adjuvants do not improve vaccine immunogenicity for influenza subunit vaccines, whereas oil-in-water adjuvants are effective, especially with H5N1-inactivated vaccines. We used whole-genome-fragment phage display libraries followed by surface plasmon resonance (SPR) technologies to elucidate the effect of different adjuvants on the antibody repertoire against H5N1 vaccine in humans. The oil-in-water adjuvant MF59 induced epitope spreading from HA2 to HA1 in hemagglutinin (HA) and neuraminidase relative to unadjuvanted or aluminum-adjuvanted vaccines. Moreover, we observed an increase by a factor of 20 in the frequency of HA1-to-HA2–specific phage clones in sera after MF59-adjuvanted vaccine administration and a factor of 2 to 3 increase in the avidity of antibodies binding to properly folded HA1(28–319), as measured by SPR. The adjuvant-dependent increase in binding to conformational HA1 epitopes correlated with broadening of cross-clade neutralization and predicted improved in vivo protection. Thus, MF59 adjuvant improves the immune response to a H5N1 vaccine by inducing qualitative and quantitative expansion of the antibody repertoires with protective potential.

Influenza vaccine immunology.

Summary:  Studying the spread of influenza in human populations and protection by influenza vaccines provides important insights into immunity against influenza. The 2009 H1N1 pandemic has taught the most recent lessons. Neutralizing and receptor‐blocking antibodies against hemagglutinin are the primary means of protection from the spread of pandemic and seasonal strains. Anti‐neuraminidase antibodies seem to play a secondary role. More broadly cross‐reactive forms of immunity may lessen disease severity but are insufficient to prevent epidemic spread. Priming by prior exposure to related influenza strains through infection or immunization permits rapid, potent antibody responses to immunization. Priming is of greater importance to the design of immunization strategies than the immunologically fascinating phenomenon of dominant recall responses to previously encountered strains (original antigenic sin). Comparisons between non‐adjuvanted inactivated vaccines and live attenuated vaccines demonstrate that both can protect, with some advantage of live attenuated vaccines in children and some advantage of inactivated vaccines in those with multiple prior exposures to influenza antigens. The addition of oil‐in‐water emulsion adjuvants to inactivated vaccines provides enhanced functional antibody titers, greater breadth of antibody cross‐reactivity, and antigen dose sparing. The MF59 adjuvant broadens the distribution of B‐cell epitopes recognized on HA and NA following immunization.

Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen

Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen–antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å2 on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen–antibody interfaces are required to understand and design effective immunogens.

Making Friends in Out-of-the- Way Places: How Cells of the Immune System Get Together and How They Conduct Their Business as Revealed by Intravital Imaging

Summary: A central characteristic of the immune system is the constantly changing location of most of its constituent cells. Lymphoid and myeloid cells circulate in the blood, and subsets of these cells enter, move, and interact within, then leave organized lymphoid tissues. When inflammation is present, various hematopoietic cells also exit the vasculature and migrate within non‐lymphoid tissues, where they carry out effector functions that support host defense or result in autoimmune pathology. Effective innate and adaptive immune responses involve not only the action of these individual cells but also productive communication among them, often requiring direct membrane contact between rare antigen‐specific or antigen‐bearing cells. Here, we describe our ongoing studies using two‐photon intravital microscopy to probe the in situ behavior of the cells of the immune system and their interactions with non‐hematopoietic stromal elements. We emphasize the importance of non‐random cell migration within lymphoid tissues and detail newly established mechanisms of traffic control that operate at multiple organizational scales to facilitate critical cell contacts. We also describe how the methods we have developed for imaging within lymphoid sites are being applied to other tissues and organs, revealing dynamic details of host‐pathogen interactions previously inaccessible to direct observation.

Human circulating influenza-CD4+ ICOS1+IL-21+ T cells expand after vaccination, exert helper function, and predict antibody responses

Protection against influenza is mediated by neutralizing antibodies, and their induction at high and sustained titers is key for successful vaccination. Optimal B cells activation requires delivery of help from CD4+ T lymphocytes. In lymph nodes and tonsils, T-follicular helper cells have been identified as the T cells subset specialized in helping B lymphocytes, with interleukin-21 (IL-21) and inducible costimulatory molecule (ICOS1) playing a central role for this function. We followed the expansion of antigen-specific IL-21+ CD4+ T cells upon influenza vaccination in adults. We show that, after an overnight in vitro stimulation, influenza-specific IL-21+ CD4+ T cells can be measured in human blood, accumulate in the CXCR5−ICOS1+ population, and increase in frequency after vaccination. The expansion of influenza-specific ICOS1+IL-21+ CD4+ T cells associates with and predicts the rise of functionally active antibodies to avian H5N1. We also show that blood-derived CXCR5−ICOS1+ CD4+ T cells exert helper function in vitro and support the differentiation of influenza specific B cells in an ICOS1- and IL-21–dependent manner. We propose that the expansion of antigen-specific ICOS1+IL-21+ CD4+ T cells in blood is an early marker of vaccine immunogenicity and an important immune parameter for the evaluation of novel vaccination strategies.

Generating memory with vaccination

The goal of vaccination is to induce long‐lasting protective immune memory. Although most vaccines induce good memory responses, the type of memory induced by different vaccines may be considerably different. In addition, memory responses to the same vaccine may be influenced by age, environmental and genetic factors. Results emerging from detailed and integrated profiling of immune‐responses to natural infection or vaccination suggest that the type and duration of immune memory are largely determined by the magnitude and complexity of innate immune signals that imprint the acquired immune primary responses. Here we summarize results obtained from analyzing human immune memory responses to different types of vaccines. We will also discuss how extending clinical investigation to events occurring early after vaccination can help identify early predictive markers of protective memory and thus contribute to faster development of better and safer vaccines.