Strength Of Immune Response Research Articles

Knockdown of swine leukocyte antigen expression in porcine lung transplants enables graft survival without immunosuppression.

Immune rejection remains the major obstacle to long-term survival of allogeneic lung transplants. The expression of major histocompatibility complex molecules and minor histocompatibility antigens triggers allogeneic immune responses that can lead to allograft rejection. Transplant outcomes therefore depend on long-term immunosuppression, which is associated with severe side effects. To address this problem, we investigated the effect of genetically engineered transplants with permanently down-regulated swine leukocyte antigen (SLA) expression to prevent rejection in a porcine allogeneic lung transplantation (LTx) model. Minipig donor lungs with unmodified SLA expression (control group, n = 7) or with modified SLA expression (treatment group, n = 7) were used to evaluate the effects of SLA knockdown on allograft survival and on the nature and strength of immune responses after terminating an initial 4-week period of immunosuppression after LTx. Genetic engineering to down-regulate SLA expression was achieved during ex vivo lung perfusion by lentiviral transduction of short hairpin RNAs targeting mRNAs encoding β2-microglobulin and class II transactivator. Whereas all grafts in the control group were rejected within 3 months, five of seven animals in the treatment group maintained graft survival without immunosuppression during the 2-year monitoring period. Compared with controls, SLA-silenced lung recipients had lower donor-specific antibodies and proinflammatory cytokine concentrations in the serum. Together, these data demonstrate a survival benefit of SLA-down-regulated lung transplants in the absence of immunosuppression.

Tumor-immune metaphenotypes orchestrate an evolutionary bottleneck that promotes metabolic transformation.

Metabolism plays a complex role in the evolution of cancerous tumors, including inducing a multifaceted effect on the immune system to aid immune escape. Immune escape is, by definition, a collective phenomenon by requiring the presence of two cell types interacting in close proximity: tumor and immune. The microenvironmental context of these interactions is influenced by the dynamic process of blood vessel growth and remodelling, creating heterogeneous patches of well-vascularized tumor or acidic niches. Here, we present a multiscale mathematical model that captures the phenotypic, vascular, microenvironmental, and spatial heterogeneity which shapes acid-mediated invasion and immune escape over a biologically-realistic time scale. The model explores several immune escape mechanisms such as i) acid inactivation of immune cells, ii) competition for glucose, and iii) inhibitory immune checkpoint receptor expression (PD-L1). We also explore the efficacy of anti-PD-L1 and sodium bicarbonate buffer agents for treatment. To aid in understanding immune escape as a collective cellular phenomenon, we define immune escape in the context of six collective phenotypes (termed "meta-phenotypes"): Self-Acidify, Mooch Acid, PD-L1 Attack, Mooch PD-L1, Proliferate Fast, and Starve Glucose. Fomenting a stronger immune response leads to initial benefits (additional cytotoxicity), but this advantage is offset by increased cell turnover that leads to accelerated evolution and the emergence of aggressive phenotypes. This creates a bimodal therapy landscape: either the immune system should be maximized for complete cure, or kept in check to avoid rapid evolution of invasive cells. These constraints are dependent on heterogeneity in vascular context, microenvironmental acidification, and the strength of immune response. This model helps to untangle the key constraints on evolutionary costs and benefits of three key phenotypic axes on tumor invasion and treatment: acid-resistance, glycolysis, and PD-L1 expression. The benefits of concomitant anti-PD-L1 and buffer treatments is a promising treatment strategy to limit the adverse effects of immune escape.

Optimization of Capture ELISAs for Chicken Cytokines Using Commercially Available Antibodies.

Cytokines like interferon (IFN)-γ, interleukin (IL)-2, IL-6, IL-10, and IL-12p40 are important biomarkers for characterizing the nature and strength of immune responses. It is important to be able to quantify the cytokines at the protein level in biological samples. Quantification of chicken cytokines is generally performed on the level of messenger RNA (mRNA) by quantitative polymerase chain reaction (qPCR) because very few capture ELISAs for the quantification of chicken cytokine proteins are commercially available. Here, we describe the optimization and validation of capture ELISAs for chicken IL-2, IL-6, IL-10, IL-12p40, and IFN-γ using commercially available antibodies and reagents. First, we determined the optimal concentrations of the antibodies. We then verified the ELISAs' performance and established that the lower limit of detection (LLOD) for all cytokines was below 32 pg/mL. The ELISAs show the same binding characteristics for recombinant and native cytokines (parallelism was <15.2% CV). Values for inter-assay variation were consistently low and mostly <20% CV. Overall, the optimized capture ELISAs are sensitive (<32 pg/mL) and reliable tools to quantify chicken cytokines. These ELISAs can easily and inexpensively be utilized in any immunological lab and may therefore have wide applicability in immunological research for poultry.

New Hopes in Vaccine Technology: mRNA Vaccines

The major disadvantages of traditional virus vaccines are time constraints in development and difficulties in large-scale production. Therefore, there is a need to develop stronger and more versatile vaccine platforms. mRNA vaccines constitute a promising alternative to traditional vaccine approaches due to their high potential, rapid development capacity, low cost production and safe administration potential. Stability and translation of mRNA are crucial for a successful RNA vaccine. It is critical to determine mRNA purity, stability, and protein yield during the translation process. Therefore, engineering the RNA sequence, such as modification of the 5' cap structure, extension of the poly (A) tail, editing nucleotide sequences in non-coding (UTR) and coding (ORF) regions, or incorporating modified nucleotides into the structure, makes synthetic mRNA more translatable. Two classes of non-replicating and self-amplifying mRNA are used as vaccines. While non-replicating mRNA only encodes protein antigens of interest, self-amplifying mRNA also encodes proteins required for RNA replication. The transfer and formulation of mRNA vaccines to cells is crucial for determining the kinetics of antigen expression, protein amount, and strength of immune response. In order to achieve this success, mRNA vaccines are given to cells in various formats such as lipid nanoparticles, polymers, peptides, and naked mRNA to develop the most effective transfer material. Recent technological advances have eliminated the low efficiency in in vivo transfer and translation and have made this vaccine platform widespread in preclinical and clinical trials against various infectious diseases and cancers. Over the past decade, major technological innovations have made mRNA a promising therapeutic tool in the fields of vaccine development and protein replacement therapy. Nowadays, antigens, neutralizing antibodies and proteins with immunostimulating activity have become coded by mRNA vaccines. Although many mRNA vaccines appear to be effective in preclinical and clinical studies, there are still some issues to be improved in terms of transfer efficiency, targeting to specific cell types, and the reliability of transfer devices. In this review, the latest developments and current challenges in the optimization, formulation and transfer of mRNA vaccines to cells with future development perspectives have been reviewed.

PRECLINICAL STUDIES AND CLINICAL TRIALS IN DETERMINATION OF SAFETY AND EFFICACY OF MENINGOCOCCAL VACCINES: PRESENT SCENARIO

Meningitis, a serious communicable inflammatory disease continues to be a worldwide threat especially in sub-Saharan Africa affecting millions of people with high death rates every year. Strains from six serogroups of the principal causative organism Neisseria meningitidis are found to be responsible for the majority of infections. Incidences of antibiotic resistance and efforts to provide mass protection have necessitated the development of meningococcal vaccines since the latter half of the 20th century. Aiming to ostracize meningitis by 2030, the World Health Organization focuses on vaccination as an important strategy to reach the goal. Due to limited efficacy and stability issues of earlier polysaccharide and protein conjugate vaccines respectively, outer membrane vesicle (OMV) vaccines were developed. Gene manipulations have also led to the development of more efficacious tailor-made OMV vaccines due to over-expression of antigenic outer membrane proteins along with lesser pyrogenicity. Available data from preclinical studies in animal models and clinical trials, on meningococcal vaccine candidates report the strength of immune response measured by serological tests such as enzyme-linked immunosorbent assay and serum bactericidal assay. Post-immunization adverse reactions have been also monitored as a part of safety assessment. The novelty of the present review lies in summarizing the outcomes of the preclinical animal studies and clinical trials conducted on various types of meningococcal vaccines till date and thereby highlighting the paucities in the existing information which can facilitate understanding the present scenario, challenges, and future scope in the field of meningococcal vaccine development.

Socioeconomic position, immune function, and its physiological markers

The development of costly traits such as immune function and secondary sexual traits is constrained by resource availability. The quality of developmental conditions and the availability of resources in ontogeny may therefore influence immune system functions and other biological traits. We analyzed causal pathways between family socioeconomic position, strength of immune response, and five physiological biomarkers in young Latvian men (n = 93) using structural equation modeling. Men from wealthier families had higher testosterone levels (rs = 0.280), stronger immune response (rs = 0.551), and higher facial attractiveness (rs = 0.300). There were weak, non-significant correlations between family income, body fat percentage (rs = −0.147), and fluctuating asymmetry (rs = −0.159). Testosterone partially (33.8%) mediated the effect of family income on facial masculinity. Testosterone (positively) and adiposity (negatively) partially (4%) mediated the relationship between family income and immune function. Higher facial masculinity, higher facial symmetry, and lower adiposity were reliable and independent cues of better immune function (R2 = 0.238) in a larger sample of young Latvian men (N = 146). Resource availability in ontogeny has an important role for the development of immune function and physical appearance, and it is a key parameter to be included in human eco-immunological research.

Circadian immune circuits.

Immune responses are gated to protect the host against specific antigens and microbes, a task that is achieved through antigen- and pattern-specific receptors. Less appreciated is that in order to optimize responses and to avoid collateral damage to the host, immune responses must be additionally gated in intensity and time. An evolutionary solution to this challenge is provided by the circadian clock, an ancient time-keeping mechanism that anticipates environmental changes and represents a fundamental property of immunity. Immune responses, however, are not exclusive to immune cells and demand the coordinated action of nonhematopoietic cells interspersed within the architecture of tissues. Here, we review the circadian features of innate immunity as they encompass effector immune cells as well as structural cells that orchestrate their responses in space and time. We finally propose models in which the central clock, structural elements, and immune cells establish multidirectional circadian circuits that may shape the efficacy and strength of immune responses and other physiological processes.

Modeling within-host viral dynamics: The role of CTL immune responses in the evolution of drug resistance

To study the emergence and evolution of drug resistance during treatment of HIV infection, we study a mathematical model with two strains, one drug-sensitive and the other drug-resistant, by incorporating cytotoxic T lymphocyte (CTL) immune response. The reproductive numbers for each strain with and without the CTL immune response are obtained and shown to determine the stability of the steady states. By sensitivity analysis, we evaluate how the changes of parameters influence the reproductive numbers. The model shows that CTL immune response can suppress the development of drug resistance. There is a dynamic relationship between antiretroviral drug administration, the prevalence of drug resistance, the total level of viral production, and the strength of immune responses. We further investigate the scenario under which the drug-resistant strain can outcompete the wild-type strain. If drug efficacy is at an intermediate level, the drug-resistant virus is likely to arise. The slower the immune response wanes, the slower the drug-resistant strain grows. The results suggest that immunotherapy that aims to enhance immune responses, combined with antiretroviral drug treatment, may result in a functional control of HIV infection.

Women\u2019s socioeconomic position in ontogeny is associated with improved immune function and lower stress, but not with height

Immune function, height and resource accumulation comprise important life history traits in humans. Resource availability models arising from life history theory suggest that socioeconomic conditions influence immune function, growth and health status. In this study, we tested whether there are associations between family income during ontogeny, adult height, cortisol level and immune response in women. A hepatitis B vaccine was administered to 66 young Latvian women from different socioeconomic backgrounds, and blood samples were then collected to measure the level of antibodies that the women produced in response to the vaccination. Cortisol levels were measured from plasma samples pre- and post-vaccination. Women from wealthier families had lower cortisol levels, and women from the highest family income group had the highest levels of antibody titers against hepatitis B vaccine. No significant relationships were observed between cortisol level and immune function, nor between family income and height. The results show that income level during ontogeny is associated with the strength of immune response and with psychoneuroendocrine pathways underlying stress perception in early adulthood. The findings indicate that the quality of the developmental niche is associated with the condition-dependent expression of immune function and stress response.

Tissue regulatory T cells.

SummaryFoxp3+ CD4+ regulatory T cells (Tregs) are an immune cell lineage endowed with immunosuppressive functionality in a wide array of contexts, including both anti‐pathogenic and anti‐self responses. In the past decades, our understanding of the functional diversity of circulating or lymphoid Tregs has grown exponentially. Only recently, the importance of Tregs residing within non‐lymphoid tissues, such as visceral adipose tissue, muscle, skin and intestine, has been recognized. Not only are Tregs critical for influencing the kinetics and strength of immune responses, but the regulation of non‐immune or parenchymal cells, also fall within the purview of tissue‐resident or infiltrating Tregs. This review focuses on providing a systematic and comprehensive comparison of the molecular maintenance, local adaptation and functional specializations of Treg populations operating within different tissues.

To Induce Immune Reconstitution Inflammatory Syndrome or Suppress It: The Spectrum of Mycobacterium genavense in the Antiretroviral Era.

Mycobacterium genavense is a challenging opportunistic pathogen to diagnose and manage in patients with human immunodeficiency virus (HIV). Persistent immunosuppression or protracted immune reconstitution inflammatory syndrome can lead to complicated clinical courses. We describe 3 cases of M. genavense in patients with HIV representing the spectrum between disease burden and strength of immune response.

Immune and environment-driven gene expression during invasion: An eco-immunological application of RNA-Seq.

Host–pathogen associations change rapidly during a biological invasion and are predicted to impose strong selection on immune function. It has been proposed that the invader may experience an abrupt reduction in pathogen‐mediated selection (“enemy release”), thereby favoring decreased investment into “costly” immune responses. Across plants and animals, there is mixed support for this prediction. Pathogens are not the only form of selection imposed on invaders; differences in abiotic environmental conditions between native and introduced ranges are also expected to drive rapid evolution. Here, we use RNA‐Seq to assess the expression patterns of immune and environmentally associated genes in the cane toad (Rhinella marina) across its invasive Australian range. Transcripts encoding mediators of costly immune responses (inflammation, cytotoxicity) showed a curvilinear relationship with invasion history, with highest expression in toads from oldest and newest colonized areas. This pattern is surprising given theoretical expectations of density dynamics in invasive species and may be because density influences both intraspecific competition and parasite transmission, generating conflicting effects on the strength of immune responses. Alternatively, this expression pattern may be the result of other evolutionary forces, such as spatial sorting and genetic drift, working simultaneously with natural selection. Our findings do not support predictions about immune function based on the enemy release hypothesis and suggest instead that the effects of enemy release are difficult to isolate in wild populations, especially in the absence of information regarding parasite and pathogen infection. Additionally, expression patterns of genes underlying putatively environmentally associated traits are consistent with previous genetic studies, providing further support that Australian cane toads have adapted to novel abiotic challenges.

To Cure or Not to Cure: Consequences of Immunological Interactions in CML Treatment.

Recent clinical findings in chronic myeloid leukemia (CML) patients suggest that the number and function of immune effector cells are modulated by tyrosine kinase inhibitors (TKI) treatment. There is further evidence that the success or failure of treatment cessation at least partly depends on the patients immunological constitution. Here, we propose a general ODE model to functionally describe the interactions between immune effector cells with leukemic cells during the TKI treatment of CML. In total, we consider 20 different sub-models, which assume different functional interactions between immune effector and leukemic cells. We show that quantitative criteria, which are purely based on the quality of model fitting, are not able to identify optimal models. On the other hand, the application of qualitative criteria based on a dynamical system framework allowed us to identify nine of those models as more suitable than the others to describe clinically observed patterns and, thereby, to derive conclusion about the underlying mechanisms. Additionally, including aspects of early CML onset, we can demonstrate that certain critical parameters, such as the strength of immune response or leukemia proliferation rate, need to change during CML growth prior to diagnosis, leading to bifurcations that alter the attractor landscape. Finally, we show that the crucial parameters determining the outcome of treatment cessation are not identifiable with tumor load data only, thereby highlighting the need to measure immune cell number and function to properly derive mathematical models with predictive power.

Beneficial Effects of Tamoxifen Treatment in Type 1 Diabetes

Type 1 diabetes (T1D) results from the destruction of pancreatic β-cell through the β-cell specific autoimmune process. β-cell autoantigens, macrophages, dendritic cells (DC), B lymphocytes, and T lymphocytes have been implicated in the pathogenesis of autoimmune diabetes. Tamoxifen is a selective estrogen receptor modulators (SERM), used in the treatment of breast cancer. Research reports have shown that tamoxifen might decrease the strength of immune responses through modulation of DC differentiation and activation, and it may depress immunity. In NOD/ShiLtJ mice (female), which is a polygenic model for T1D, we found that tamoxifen exerts antidiabetic effects through acting on the immune system, leading to the suppression of insulitis and systemic inflammatory cytokine production in the in-vivo model. Tamoxifen therapy preserved the β-cell mass and prevented the development of T1D. Tamoxifen providing β-cell protection and immunoinhibitory effects offer a novel insight into the possible role of tamoxifen in the regulation of islet cell function and glucose homeostasis. These findings are significant and useful for clinical application. Disclosure J. Zheng: None. M. Zafar: None. L. Chen: None. X. Guo: None. X. Li: None.

Mitogen-activated protein kinase eight polymorphisms are associated with immune responsiveness to HBV vaccinations in infants of HBsAg(+)/HBeAg(\u2212) mothers

BackgroundInfants born to hepatitis B surface antigen (HBsAg) positive mothers are at a higher risk for Hepatitis B virus (HBV) infection. Host genetic background plays an important role in determining the strength of immune response to vaccination. We conducted this study to investigate the association between Tumor necrosis factor (TNF) and Mitogen-activated protein kinase eight (MAPK8) polymorphisms and low response to hepatitis B vaccines.MethodsA total of 753 infants of HBsAg positive and hepatitis Be antigen (HBeAg) negative mothers from the prevention of mother-to-infant transmission of HBV cohort were included. Five tag single nucleotide polymorphism (SNPs) (rs1799964, rs1800629, rs3093671, rs769177 and rs769178) in TNF and two tag SNPs (rs17780725 and rs3827680) in MAPK8 were genotyped using the MassARRAY platform.ResultsA higher percentage of breastfeeding (P = 0.013) and a higher level of Ab titers were observed in high responders (P < 0.001). The MAPK8 rs17780725 AA genotype increased the risk of low response to hepatitis B vaccines (OR = 3.176, 95% CI: 1.137–8.869). Additionally, subjects with the AA genotype may have a lower Ab titer than subjects with GA or GG genotypes (P = 0.051). Compared to infants who were breastfed, infants who were not breastfed had an increased risk of low response to hepatitis B vaccine (OR = 2.901, 95% CI:1.306–6.441).ConclusionsMAPK8 polymorphisms are associated with immune response to HBV vaccinations in infants of HBsAg(+)/HBeAg(−) mothers.

Tumor mutation burden to tumor burden ratio and prediction of clinical benefit of anti-PD-1/PD-L1 immunotherapy

Immune checkpoint inhibitors have profoundly altered the therapeutic landscape of several malignancies. The establishment of predictive biomarkers for checkpoint blockades is of the considerable importance in the identification of populations likely to experience a good response to immunotherapy and to maximize the therapeutic benefits. Several trials showed that the tumor mutation burden (TMB) could predict the response to immunotherapy, but some lower clinical benefit was also seen in cancer with high TMB. The imbalance between the strength of immune response and pretreatment tumor burden (TB) could also cause immunotherapy to fail in cancer patients. For this reason, we hypothesized that the TMB-TB ratio could predict the clinical benefit of checkpoint inhibitor immunotherapy and that PFS or ORRs should be used more often in patients with high TMB-TB ratio than in individuals with low TMB-TB ratios.

Alternative polyadenylation signals regulate HLA-A surface expression

Abstract Genomic variations in the 5′ and 3′ untranslated region (UTR) have been shown to influence Human leukocyte antigen (HLA) class I expression, the strength of immune responses and disease outcomes beyond peptide binding. Sequencing of the 3′UTR of common HLA-A alleles indicated a presence of three polyadenylation signals (PAS). The proximal and the middle PAS are conserved whereas the distal PAS is disrupted in some alleles. Using 3′ rapid amplification of cDNA ends (3′RACE), we confirmed expression of two distinct forms of HLA-A 3′UTR that use the proximal or the distal PAS and differ in length by 100 base pairs. HLA-A alleles varied in the usage of PAS, with some alleles using only the proximal, and others using both the PAS to differing degrees. The short and long 3′UTRs cloned in luciferase vectors and expressed in the human T cell line-Jurkat showed similar mRNA expression levels. However, the long 3′UTR conferred lower luciferase expression as compared to the short form, indicating translation inhibition of the long 3′UTR. RNA affinity pulldown followed by mass-spectrometric analysis indicated enrichment of an RNA-binding protein (RBP), Syncrip, among proteins bound to the long 3′UTR. Only the long form of cellular HLA-A 3′UTR co-precipitated with Syncrip. The siRNA depletion of Syncrip increased surface expression of HLA-A alleles with the long 3′UTR, whereas an allele expressing only the short form was unaffected. Further, specific blocking of the proximal 3′UTR reduced surface expression without a decrease in mRNA expression. These data demonstrate allele-specific variation in the PAS usage by HLA-A modulated its expression, which adds another layer of regulation and variation to the expression of HLA-A.

Immune responses link parasite genetic diversity, prevalence and plumage morphs in common buzzards

The evolution and maintenance of conspicuous phenotypic polymorphisms has challenged evolutionary ecologists for centuries. Polymorphisms in pleiotropic cascades or genetic linkage may lead to correlations of life history traits such as immunity, parasite infection levels, pigmentation and lifetime reproductive success. The common buzzard Buteo buteo is a bird of prey occurring in several plumage morphs, which differ in pigmentation and in the prevalence, infection intensity and clone composition of their most common blood parasite, as well as in ectoparasite infestation levels. Buzzard morphs are heritable and exhibit a heterozygote advantage where intermediates have higher lifetime reproductive success (LRS). We explored the hypothesis that the differences in pigmentation also correspond to differences in immunity. We hence compared an inducible adaptive and an innate constitutive immune response between the buzzard plumage morphs. The increase of specific anti-tetanus antibodies after vaccination was explained by the morph of the nestling’s mother and was highest in offspring of intermediate mothers. Additionally, nestlings with higher humoral response were less infected with blood parasites and, if infected, harboured a lower genetic diversity of these parasites. The phytohaemagglutinin-induced skin swelling, a complex of cellular inflammatory responses, was lowest in intermediate nestlings. The higher LRS of intermediate buzzards suggests that the cellular immunity is an inferior fitness determinant compared to humoral immunity. The strength of immune responses was not linear along the melanisation gradient, indicating that there is most likely no simple genetic correlation between immune responses and plumage morphs.

Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells.

Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure Lewis(X) (Le(X)) re-directs OVA to the C-type lectin receptor MGL1. Le(X)-modification of OVA favored Th1 skewing of CD4(+) T cells and enhanced cross-priming of CD8(+) T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, Le(X) modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-Le(X)-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8(+) effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-Le(X) neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11(+)LAMP1(+) compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies.

Body height, immunity, facial and vocal attractiveness in young men.

Health, facial and vocal attributes and body height of men may affect a diverse range of social outcomes such as attractiveness to potential mates and competition for resources. Despite evidence that each parameter plays a role in mate choice, the relative role of each and inter-relationships between them, is still poorly understood. In this study, we tested relationships both between these parameters and with testosterone and immune function. We report positive relationships between testosterone with facial masculinity and attractiveness, and we found that facial masculinity predicted facial attractiveness and antibody response to a vaccine. Moreover, the relationship between antibody response to a hepatitis B vaccine and body height was found to be non-linear, with a positive relationship up to a height of 188cm, but an inverse relationship in taller men. We found that vocal attractiveness was dependent upon vocal masculinity. The relationship between vocal attractiveness and body height was also non-linear, with a positive relationship of up to 178cm, which then decreased in taller men. We did not find a significant relationship between body height and the fundamental frequency of vowel sounds provided by young men, while body height negatively correlated with the frequency of second formant. However, formant frequency was not associated with the strength of immune response. Our results demonstrate the potential of vaccination research to reveal costly traits that govern evolution of mate choice in humans and the importance of trade-offs among these traits.