Cornelia L. Dekker

Variation in the human immune system is largely driven by non-heritable influences

There is considerable heterogeneity in immunological parameters between individuals, but its sources are largely unknown. To assess the relative contribution of heritable versus non-heritable factors, we have performed a systems-level analysis of 210 healthy twins between 8 and 82 years of age. We measured 204 different parameters, including cell population frequencies, cytokine responses, and serum proteins, and found that 77% of these are dominated (>50% of variance) and 58% almost completely determined (>80% of variance) by non-heritable influences. In addition, some of these parameters become more variable with age, suggesting the cumulative influence of environmental exposure. Similarly, the serological responses to seasonal influenza vaccination are also determined largely by non-heritable factors, likely due to repeated exposure to different strains. Lastly, in MZ twins discordant for cytomegalovirus infection, more than half of all parameters are affected. These results highlight the largely reactive and adaptive nature of the immune system in healthy individuals.

Cellular Immune Responses in Children and Adults Receiving Inactivated or Live Attenuated Influenza Vaccines

ABSTRACT The patterns of cellular immune responses induced by live attenuated influenza vaccine (LAIV) versus those of the trivalent inactivated influenza vaccine (TIV) have not been studied extensively, especially in children. The goals of this study were to evaluate the effects of TIV and LAIV immunization on cellular immunity to live influenza A virus in children and adults and to explore factors associated with variations in responses to influenza vaccines among individuals. A gamma interferon (IFN-γ) flow cytometry assay was used to measure IFN-γ-producing (IFN-γ+) NK and T cells in peripheral blood mononuclear cell cultures stimulated with a live influenza A virus strain before and after LAIV or TIV immunization of children and adults. The mean percentages of influenza A virus-specific IFN-γ+ CD4 and CD8 T cells increased significantly after LAIV, but not TIV, immunization in children aged 5 to 9 years. No increases in the mean levels of influenza A virus-reactive IFN-γ+ T cells and NK cells were observed in adults given LAIV or TIV. TIV induced a significant increase in influenza A virus-reactive T cells in 6-month- to 4-year-old children; LAIV was not evaluated in this age group. The postvaccination changes (n-fold) in the percentages of influenza A virus-reactive IFN-γ+ T and NK cells in adults were highly variable and correlated inversely with the prevaccination percentages, in particular with that of the CD56dim NK cell subset. In conclusion, our findings identify age, type of vaccine, and prevaccination levels of immune reactivity to influenza A virus as factors significantly associated with the magnitude of cellular immune responses to influenza vaccines.

Genetic and Environmental Determinants of Human NK Cell Diversity Revealed by Mass Cytometry

Both genetics and environment contribute to human NK cell diversity. NK Cell Nature Versus Nurture Natural killer (NK) cells were first discovered because of their ability to kill tumor cells without any previous exposure. However, this population is actually quite heterogeneous: Different subgroups of NK cells express different combinations of activating and inhibiting receptors that govern their specificity. Now, Horowitz et al. use mass cytometry to examine NK cell diversity in humans. The authors examined 35 parameters simultaneously in 5 sets of monozygotic twins as well as 12 unrelated donors. They found up to 30,000 phenotypic NK cell populations in a given individual. What’s more, by comparing the twins versus unrelated donors, they determined that although genetics primarily determined inhibitory receptor expression, activating receptors were controlled by the environment. These data suggest that inhibitory receptors may contribute more to NK cell self-tolerance, whereas activating receptors may guide response to pathogens and tumors. Natural killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 37 parameters, including 28 NK cell receptors, on peripheral blood NK cells from 5 sets of monozygotic twins and 12 unrelated donors of defined human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6000 to 30,000 phenotypic populations within an individual and >100,000 phenotypes in the donor panel. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.

Placebo-controlled trial of vaccination with recombinant glycoprotein D of herpes simplex virus type 2 for immunotherapy of genital herpes

Immunotherapy of chronic viral diseases with vaccines is an important but unproven concept. We investigated the effect of a vaccine containing recombinant glycoprotein D (gD2) of herpes simplex virus type 2 (HSV-2) on the frequency of symptomatic outbreaks in patients with genital herpes. 98 patients with documented genital herpes who reported 4-14 recurrences per year were enrolled in a double-blind, placebo-controlled trial. Subjects received injections of either 100 micrograms gD2 in alum or alum alone (placebo) at 0 and 2 months, and recurrences were documented for 1 year. The vaccine was well tolerated. gD2 recipients reported fewer recurrences per month than placebo recipients (mean 0.42 [SE 0.05] vs 0.55 [0.05]; p = 0.055), had fewer virologically confirmed recurrences per month (0.18 [0.03] vs 0.28 [0.03]; p = 0.019), and had a lower median number of recurrences for the study year (4 [range 0-17] vs 6 [0-15]; p = 0.039). Neither genital recurrence nor the placebo vaccine had any discernible effect on HSV-2-specific antibody responses, but gD2 vaccine boosted neutralising antibodies to HSV-2 fourfold and gD2-specific titres sevenfold over baseline levels. These results inspire optimism about the potential use of vaccine for the treatment of chronic, recurring viral diseases.

Lineage Structure of the Human Antibody Repertoire in Response to Influenza Vaccination

High-throughput sequencing suggests age-related differences in the changes in the human antibody repertoire after influenza vaccination. Antibodies Act Their Age One of the main advantages of the immune system in fighting infection is its ability to diversify—antibody and T cell receptor genes physically rearrange, creating a repertoire of potential responses that can be called upon and expanded if needed. However, the very diversity of this repertoire is what makes immune responses hard to study. Although we can know how any individual B cell or antibody responds to stimulation, getting the big picture is much more difficult. Adding in another variable, such as time, further complicates things. Now, Jiang et al. use high-throughput long read sequencing to characterize the human antibody response after influenza vaccination. People of age are thought to have altered immune systems compared to younger individuals. However, why exactly how the antibody repertoire changes with age remains unclear. By analyzing more than 5 million antibody heavy chain sequences, the authors were able to compare isotype diversity, lineage structure, and mutational activity in differently aged populations. They found that the elderly have fewer lineages, with reduced diversity, compared with younger subjects; however, the antibodies present before vaccination had higher levels of mutation. These same techniques can be used to study individual-specific immune responses and may aid in optimizing vaccination in the future. The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, including that each B cell’s genome encodes a distinct antibody sequence, that the antibody repertoire changes over time, and the high similarity between antibody sequences. We have addressed these challenges by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire, and measure age- and antigen-related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals’ repertoires shows that elderly subjects have a decreased number of lineages but an increased prevaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system’s clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

A Recombinant Glycoprotein Vaccine for Herpes Simplex Type 2: Safety and Efficacy

Genital infections caused by herpes simplex viruses (HSV) are prevalent worldwide [1]. The virus is the major cause of genital ulcerations in Europe and North America [2-4], and seroprevalence studies have shown that between 10% and 20% of European and North American persons aged 20 to 45 years have evidence of HSV-2 infection [1, 5, 6]. Annual acquisition rates of HSV-2 in sexually active populations are estimated to be 1% to 4% [1, 7-10]; among attendees of sexually transmitted disease clinics, the prevalence of HSV-2 seropositivity ranges from 30% to 80% [1, 7]. The incidence of neonatal herpes, one of the major complications of genital herpes, has increased two- to fivefold in the United States and Scandinavia in the last two decades [6, 11]. Vaccines for preventing or treating genital herpes would be a major public health advance. Several observations support the protective effect of preexisting HSV-specific immune responses in limiting the acquisition of HSV-2, the predominant virus type isolated from the genital tract: 1) Reinfection of persons with a second strain of the same viral subtype in the same anatomical area rarely occurs [12-15]; 2) acquisition of neonatal HSV-2 is uncommon among infants exposed to the virus at the time of delivery if the mother is HSV-2-seropositive [16, 17]; and 3) previous HSV-1 infection of the oral-labial region reduces the acquisition and severity of subsequent HSV-2 infection by 40% to 60% [7, 8, 18-22]. Neutralizing antibodies to HSV are predominantly directed to the viral surface glycoproteins, especially to the two essential and abundant glycoproteins D (gD) and B (gB) [23-25]. Monoclonal antibodies to gB and gD protect mice from experimental challenge with HSV [26, 27]. Herpes simplex virus type 2 glycoprotein D (gD2) and B (gB2) have been produced by the use of recombinant DNA technology in Chinese hamster ovary cells as carboxyl terminal-truncated derivatives [28, 29]. These recombinant proteins have been shown to protect guinea pigs from experimental genital challenge with HSV [30-33]. The extent of protection primarily depends on maintaining conformational epitopes of the protein and the potency of the adjuvant used [33, 34]. Immunization with formulations of the two recombinant proteins in the MF59 adjuvant emulsion, which contains squalene, polysorbate 80 (Tween 80, ICI America, Wilmington, Delaware), and sorbitan trioleate (Span 85, ICI America), increases humoral and cellular immune responses and induces protection from experimental challenge in animals [33-35]. Immunization with vaccines containing the recombinant proteins gB and gD combined with the MF59 adjuvant also reduces the rate of subsequent recurrences of genital herpes among previously infected guinea pigs [36, 37]. Our investigations were the initial phase I and II clinical trials to evaluate an HSV-2 subunit vaccine that contained derivatives of gB2 and gD2 in the MF59 adjuvant emulsion and was administered to persons seronegative for HSV or seropositive only for HSV-1 antibodies. These serologic groups represent the target population for a vaccine that prevents the acquisition of HSV-2, the major pathogen of genital herpes. Methods Patients We analyzed 137 patients who had no evidence of HSV-2 antibodies in their enrollment serum samples. Patients were enrolled in two phase I and two phase II clinical trials (Table 1); 65 patients lacked antibodies to both HSV-1 and HSV-2 at entry (HSV-seronegative), and 72 had only HSV-1 antibodies at entry (HSV-1-seropositive), as determined by HSV Western blot assay [7, 38]. The two initial phase I, open-label trials were done in a dose-escalating format: The first patients received 10-micrograms doses of gD2 and gB2, the second cohort received 30 g of each protein, and the last cohort received 100 g of each protein (Table 1). In the subsequent two phase II trials, patients were randomly assigned to receive one of these three doses. Because the demographic, side-effect, and immunogenicity profiles of the 23 patients enrolled in the dose-escalating trials and the 114 patients enrolled in the randomized trials were similar, the data have been combined and analyzed together. All patients were recruited from three metropolitan areas, were in excellent health without chronic disease or evidence of genital HSV-2 infection, and were not receiving long-term immunosuppressive therapy or acyclovir. The 114 patients enrolled in the phase II trials were selected from 313 volunteers originally screened for the trial, of whom 83 were rejected because they had HSV-2 antibodies. Fifty of the 67 patients seronegative for HSV and 64 of the 163 patients seropositive for HSV-1 were enrolled. Vaccine was administered intramuscularly into the deltoid at 0, 1, and 6 months. Serum samples for HSV-2 antibody assays were obtained before enrollment and at study days 0 (before immunization), 28, 42, 180, 194, 270, and 360. All patients were examined in the clinic for 30 minutes after each injection and again 24 hours after injection. In addition, patients were given diary cards to record local symptoms (pain, erythema, and induration), constitutional symptoms (headache, fever, malaise, and myalgia), and any disruption of activities on a daily basis for the 7 days after each injection. Table 1. Patient Enrollment in Phase I and II Trials of a Recombinant HSV-2 Vaccine Containing Glycoproteins gB2 and gD2* Side effects were classified as mild, moderate, or severe according to the following definitions: mild = transient, with no limitation in activity; moderate = moderate effect on the patients daily activity; and severe = medical intervention required, with a marked effect on patients daily activity. Occurrences of erythema or induration were recorded as 0 to 10 mm, greater than 10 to 30 mm, greater than 30 to 50 mm, and greater than 50 mm on the basis of the maximal diameter measured during the visits or from diary cards for the 7-day observation period after each injection. The maximum severity of each symptom recorded during this 7-day period is similarly presented. Blood was drawn on days 0, 1, and 14 for the assessment of serum chemistries, liver functions, leukocyte counts, and erythrocyte sedimentation rates. A single lot of vaccine was used for each antigen dose for all vaccinations. The protocol was approved by the institutional review board at each institution, and each patient signed an informed consent form for the screening and vaccination protocol regimen. Patients were monetarily compensated for their time and were instructed to avoid sexual exposure to genital lesions and persons with HSV-2 infection. To compare the HSV gB2, gD2, and neutralizing antibody titers in vaccine recipients with those found in naturally infected persons, we analyzed these HSV antibody responses in unimmunized persons whose HSV Western blot results indicated the presence of only HSV-1 antibodies (n = 249) or only HSV-2 antibodies (n = 133). These persons were being screened for participation in other vaccine trials. Ninety-eight (74%) of the 133 persons seropositive for HSV-2 had frequently recurring (4 to 14 episodes per year), culture-proven, symptomatic genital HSV-2 infection [39]. We did not collect information on the symptoms of the remaining 35 patients seropositive for HSV-2 and the 249 patients seropositive for HSV-1. Thus, the naturally infected comparison group represented a cross-section of persons with subclinical and clinical HSV-1 and HSV-2 infection. Because neutralizing antibodies to HSV-2 have not been found to vary with symptomatic reactivations and because the HSV-2 titers of the 98 symptomatic and the 35 other HSV-2-seropositive patients did not differ, we used the initial serum sample that was collected for assaying the gB2, gD2, and HSV neutralizing antibodies among the naturally infected cohort [39, 40]. Laboratory Studies Antibodies to HSV-2 gB and gD were measured by an enzyme-linked immunosorbent assay (ELISA) as previously described [41]. In brief, 50 L of recombinant proteins diluted to 6 g/mL were separately dispensed into 96-well microtiter plates and incubated for 1 hour at 37 C. Unbound antigen was removed by washing. Serum samples were initially diluted 1:27, the lowest dilution that gave no background value in seronegative patients. Serial dilutions that increased threefold from 1:27 to 1:59 049 were made in the plate, and the plates were incubated for 1 hour at 37 C. Unbound antibodies were removed by washing, and bound antibody-antigen complexes were detected by goat antihuman IgG conjugated to horseradish peroxidase, with subsequent colorimetric development with o-phenylenediamine dihydrochloride (Sigma, St. Louis, Missouri) as the substrate. Optical densities were read at 490 nm with a Titertek spectrophotometer (Flow Laboratories, McClean, Virginia), and titers were determined as the reciprocal of the dilution at which an optical density of 1.0 was reached. A standard human HSV-positive serum sample (Boston Biomedica, Inc., West Bridgewater, Massachusetts) was included on each plate, and the final titer was adjusted so that it matched the standard curve. All serum samples were run in duplicate against both antigens, and reported titers represent the average of the two assay points. In addition, a second internal standard was run on each plate to confirm the internal consistency of the assay. The coefficients of variation for the gB2 and gD2 assays were approximately 34% and 30%, respectively. Neutralizing HSV-2 antibody titers were measured with a complement-dependent microneutralization assay. Serum samples were diluted with a twofold serial dilution and then combined with 1.7 102 plaque-forming units of HSV-2 strain 333 [42] and an equal volume of guinea pig complement at a 1:125 dilution (Gibco BRL, Gaithersburg, Maryland) for 2 hours at 37 C. The mixture was subsequently transferred to a 96-well microtiter plate that contained confluent Vero cell mo

Effects of Adjuvants on the Safety and Immunogenicity of an Avian Influenza H5N1 Vaccine in Adults

BACKGROUND Influenza A H5N1 viruses pose a significant threat to human health. METHODS We conducted a multicenter, randomized, double-blind study in 394 healthy adults. Subjects were randomly assigned to receive 2 intramuscular doses of either saline placebo; influenza A/Vietnam/1203/2004(H5N1) vaccine alone at 45, 30, or 15 microg per dose; vaccine at 15 or 7.5 microg per dose with MF59; or vaccine at 30, 15, or 7.5 microg per dose with aluminum hydroxide. Subjects were followed up for safety and blood samples were obtained to determine antibody responses. RESULTS The vaccine formulations were well tolerated but local adverse effects were common; the incidence of these effects increased in a dose-dependent manner and was increased by the addition of adjuvants. The addition of MF59 increased the antibody response, whereas the addition of aluminum hydroxide did not. The highest antibody responses were seen in the group that received 15 microg of vaccine per dose with MF59, in which 63% of subjects achieved the predetermined endpoint (hemagglutination-inhibition titer > or =40) 28 days after the second dose, compared with 29% in the group that received the highest dose (45 microg per dose) of vaccine alone. CONCLUSIONS A 2-dose regimen of subvirion influenza A (H5N1) vaccine was well tolerated. The antibody responses to 15 microg of A/H5 vaccine with MF59 were higher than the responses to 45 microg of vaccine alone. TRIAL REGISTRATION ClincalTrials.gov identifier: http://www.clinicaltrials.gov/ct2/show/NCT00280033?term= NCT00280033&rank=1 NCT00280033 .

Systems analysis of sex differences reveals an immunosuppressive role for testosterone in the response to influenza vaccination

Significance There are marked differences between the sexes in their immune response to infections and vaccination, with females often having significantly higher responses. However, the mechanisms underlying these differences are largely not understood. Using a systems immunology approach, we have identified a cluster of genes involved in lipid metabolism and likely modulated by testosterone that correlates with the higher antibody-neutralizing response to influenza vaccination observed in females. Moreover, males with the highest testosterone levels and expression of related gene signatures exhibited the lowest antibody responses to influenza vaccination. This study generates a number of hypotheses on the sex differences observed in the human immune system and their relationship to mechanisms involved in the antibody response to vaccination. Females have generally more robust immune responses than males for reasons that are not well-understood. Here we used a systems analysis to investigate these differences by analyzing the neutralizing antibody response to a trivalent inactivated seasonal influenza vaccine (TIV) and a large number of immune system components, including serum cytokines and chemokines, blood cell subset frequencies, genome-wide gene expression, and cellular responses to diverse in vitro stimuli, in 53 females and 34 males of different ages. We found elevated antibody responses to TIV and expression of inflammatory cytokines in the serum of females compared with males regardless of age. This inflammatory profile correlated with the levels of phosphorylated STAT3 proteins in monocytes but not with the serological response to the vaccine. In contrast, using a machine learning approach, we identified a cluster of genes involved in lipid biosynthesis and previously shown to be up-regulated by testosterone that correlated with poor virus-neutralizing activity in men. Moreover, men with elevated serum testosterone levels and associated gene signatures exhibited the lowest antibody responses to TIV. These results demonstrate a strong association between androgens and genes involved in lipid metabolism, suggesting that these could be important drivers of the differences in immune responses between males and females.

Influence of Prior Influenza Vaccination on Antibody and B-Cell Responses

Currently two vaccines, trivalent inactivated influenza vaccine (TIV) and live attenuated influenza vaccine (LAIV), are licensed in the USA. Despite previous studies on immune responses induced by these two vaccines, a comparative study of the influence of prior influenza vaccination on serum antibody and B-cell responses to new LAIV or TIV vaccination has not been reported. During the 2005/6 influenza season, we quantified the serum antibody and B-cell responses to LAIV or TIV in adults with differing influenza vaccination histories in the prior year: LAIV, TIV, or neither. Blood samples were collected on days 0, 7–9 and 21–35 after immunization and used for serum HAI assay and B-cell assays. Total and influenza-specific circulating IgG and IgA antibody secreting cells (ASC) in PBMC were detected by direct ELISPOT assay. Memory B cells were also tested by ELISPOT after polyclonal stimulation of PBMC in vitro. Serum antibody, effector, and memory B-cell responses were greater in TIV recipients than LAIV recipients. Prior year TIV recipients had significantly higher baseline HAI titers, but lower HAI response after vaccination with either TIV or LAIV, and lower IgA ASC response after vaccination with TIV than prior year LAIV or no vaccination recipients. Lower levels of baseline HAI titer were associated with a greater fold-increase of HAI titer and ASC number after vaccination, which also differed by type of vaccine. Our findings suggest that the type of vaccine received in the prior year affects the serum antibody and the B-cell responses to subsequent vaccination. In particular, prior year TIV vaccination is associated with sustained higher HAI titer one year later but lower antibody response to new LAIV or TIV vaccination, and a lower effector B-cell response to new TIV but not LAIV vaccination.

Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies

During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies.