Sex-specific aspects in patients with oropharyngeal squamous cell carcinoma: a bicentric cohort study.

BACKGROUND: Oropharyngeal squamous cell carcinoma (OPSCC) is the only subgroup of head neck cancer that presents with an increased incidence. Gender-specific studies in other cancer entities have revealed differences in treatment response and prognosis. However, only limited data in OPSCC according to gender and human papillomavirus (HPV) status exist. Therefore, we aimed to investigate sex-specific differences in OPSCC and how these may be distributed in relation to HPV and other risk factors. METHODS: This retrospective, bicentric study included 1629 patients with OPSCC diagnosed between 1992 and 2020. We formed subgroups based on TNM status, American Joint Cancer Committee 8th edition (AJCC8), HPV status, treatment modality (surgery (± radio(chemo)therapy (RCT) vs. definitive RCT) and patient-related risk factors and investigated gender differences and their impact on patients survival via descriptive-,uni- and multivariate analysis. RESULTS: With the exception of alcohol abuse, no significant differences were found in risk factors between men and women. Females presented with better OS than males in the subgroup T1-2, N + , independent of risk factors (p = 0.008). Males demonstrated significant stratification through all AJCC8 stages (all p < 0.050). In contrast, women were lacking significance between stage II and III (p = 0.992). With regard to therapy (surgery (± R(C)T) - vs. definitive RCT) women treated with surgery had better OS than men in the whole cohort (p = 0.008). Similar results were detected in the HPV-negative OPSCC sub-cohort (p = 0.042) and in high-risk groups (AJCC8 stage III and IV with M0, p = 0.003). CONCLUSION: Sex-specific differences in OPSCC represent a health disparity, particularly according to staging and treatment, which need to be addressed in future studies.

Predicting HPV association using deep learning and regular H&E stains allows granular stratification of oropharyngeal cancer patients.

Human Papilloma Virus (HPV)-associated oropharyngeal squamous cell cancer (OPSCC) represents an OPSCC subgroup with an overall good prognosis with a rising incidence in Western countries. Multiple lines of evidence suggest that HPV-associated tumors are not a homogeneous tumor entity, underlining the need for accurate prognostic biomarkers. In this retrospective, multi-institutional study involving 906 patients from four centers and one database, we developed a deep learning algorithm (OPSCCnet), to analyze standard H&E stains for the calculation of a patient-level score associated with prognosis, comparing it to combined HPV-DNA and p16-status. When comparing OPSCCnet to HPV-status, the algorithm showed a good overall performance with a mean area under the receiver operator curve (AUROC) = 0.83 (95% CI = 0.77-0.9) for the test cohort (n = 639), which could be increased to AUROC = 0.88 by filtering cases using a fixed threshold on the variance of the probability of the HPV-positive class - a potential surrogate marker of HPV-heterogeneity. OPSCCnet could be used as a screening tool, outperforming gold standard HPV testing (OPSCCnet: five-year survival rate: 96% [95% CI = 90-100%]; HPV testing: five-year survival rate: 80% [95% CI = 71-90%]). This could be confirmed using a multivariate analysis of a three-tier threshold (OPSCCnet: high HR = 0.15 [95% CI = 0.05-0.44], intermediate HR = 0.58 [95% CI = 0.34-0.98] p = 0.043, Cox proportional hazards model, n = 211; HPV testing: HR = 0.29 [95% CI = 0.15-0.54] p < 0.001, Cox proportional hazards model, n = 211). Collectively, our findings indicate that by analyzing standard gigapixel hematoxylin and eosin (H&E) histological whole-slide images, OPSCCnet demonstrated superior performance over p16/HPV-DNA testing in various clinical scenarios, particularly in accurately stratifying these patients.

Enhanced stability of the SARS CoV-2 spike glycoprotein following modification of an alanine cavity in the protein core.

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala1016 and Ala1020 in the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala1016/Ala1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID50s) in the range 2,700-5,110 for ancestral and Delta-derived viruses, and 210-1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.

Predictors for Survival of Patients with Squamous Cell Carcinoma of Unknown Primary in the Head and Neck Region.

BACKGROUND: Human papillomavirus (HPV) status is the most important predictor of survival in oropharyngeal squamous cell carcinoma (OPSCC). In patients with cervical lymph node metastases of squamous cell carcinoma of unknown origin (CUPHNSCC), much less is known. METHODS: We assessed a consecutive cohort of CUPHNSCC diagnosed from 2000-2018 for HPV DNA, mRNA, p16INK4a (p16) expression, and risk factors to identify prognostic classification markers. RESULTS: In 32/103 (31%) CUPHNSCC, p16 was overexpressed, and high-risk HPV DNA was detected in 18/32 (56.3%). This was mostly consistent with mRNA detection. In recursive partitioning analysis, CUPHNSCC patients were classified into three risk groups according to performance status (ECOG) and p16. Principal component analysis suggests a negative correlation of p16, HPV DNA, and gender in relation to ECOG, as well as a correlation between N stage, extranodal extension, and tobacco/alcohol consumption. CONCLUSIONS: Despite obvious differences, CUPHNSCC shares similarities in risk profile with OPSCC. However, the detection of p16 alone appears to be more suitable for the classification of CUPHNSCC than for OPSCC and, in combination with ECOG, allows stratification into three risk groups. In the future, additional factors besides p16 and ECOG may become important in larger studies or cases with special risk profiles.

[Salivary gland carcinomas - Monocentric experience on subtypes and their incidences over 42 years]. / Speicheldrüsenkarzinome ­ Monozentrische Erfahrung zu Subtypen und deren Inzidenz über 42 Jahre.

OBJECTIVE: Salivary gland carcinomas are rare and heterogeneous. More than 20 subtypes are recognized and risk factors are diverse. The aim of this work was to evaluate the subtype and other risk factors in a monocentric population from more than four decades. MATERIAL AND METHODS: 205 cases (diagnosis period 1972-2014) were retrospectively collected and analyzed with regard to the distribution of risk factors and their influence on overall survival (OS). RESULTS: 19/24 (79.2%) of the subtypes listed in the WHO classification occurred rarely in the cohort (< 5%). 10/24 (41.7%) of all subtypes were never diagnosed. With a total of 145/205 cases (70.7%), squamous cell carcinoma (PEC), adenocarcinoma (AdenoCa), acinar cell carcinoma (AcinarCa), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) were by far the most common subtypes. Risk factors are significantly different in these groups (e.g., lymphogenic metastasis and degree of differentiation in AdenoCa and age, T and UICC stage in PEC). The 5-year overall survival of all patients was 66.9% and differed significantly within the most common subtypes. An independent impact on overall survival was detectable for patient age (p<0.001), and T- (p=0.003) and N-stage (p=0.046) in multivariate analysis. CONCLUSIONS: Most subtypes occurred markedly rarely or not at all within decades. The most common diagnoses differ with respect to risk factors as well as OS and 3 risk groups can be defined based on histology. In conclusion, considering TNM alone is insufficient for prognosis estimation in salivary gland carcinoma.

Awareness of Human Papillomavirus (HPV) and HPV Vaccination amongst the General Population in Germany: Lack of Awareness and Need for Action.

INTRODUCTION: The oncogenic human papillomaviruses (HPV) types 16 and 18 contribute to more than 73% cases of all HPV-related cancers and commonly affect the anogenital and head and neck region, with rapidly rising incidence rates of HPV-related oropharyngeal squamous cell carcinomas (OPSCC). HPV vaccination has the potential to decrease the burden of HPV-related disease, but vaccination rates remain low in many countries. We investigated the level of awareness of HPV, and HPV-OPSCC in particular, in a representative sample of the German population. MATERIALS AND METHODS: As part of an online, population-based survey, an electronic questionnaire was administered to a representative sample of 1,095 adult individuals with a specific emphasis on awareness of HPV, transmission, and indicator symptoms of oropharyngeal cancer. Statistical analysis of levels of awareness and relation of these to age, gender, and socioeconomic background were conducted using the IBM SPSS Statistics Version 25.0. RESULTS: 699/1,095 (63.8%) subjects had never heard of HPV. Of the subjects with awareness for HPV, 210 knew that HPV could be transmitted during sex (58.3%) and 138 recognized HPV as a risk factor for OPSCC (14.2%), unrelated to gender (p = 0.357), educational status (p = 0.581), or family status (p = 0.719). 416 subjects knew that a preventive vaccine against HPV existed (44.9%). Women were significantly more aware of HPV (34.2% vs. 22.8%, p < 0.001) and the vaccination (56.4% vs. 32.7%, p < 0.001) as were men. Younger individuals (age group 25-34) were significantly more aware of HPV (p < 0.001), likely as they were offered and/or had received the HPV vaccination. There was no regional variation of HPV awareness within the German state (p = 0.051). CONCLUSION: Here we demonstrate a significant lack of awareness of HPV and HPV vaccination in a representative sample of the German population. Levels of awareness of the link of HPV and oropharyngeal cancer are particularly low, bearing in mind that this cancer is commonly affecting men and incidence rates are rapidly rising in many European countries and the USA. Awareness programs and further education are required to tackle the low awareness rates and increase the uptake of the vaccination program not only in Germany, but also worldwide.

Carbon Ion Beam Boost Irradiation in Malignant Tumors of the Nasal Vestibule and the Anterior Nasal Cavity as an Organ-Preserving Therapy.

BACKGROUND: Surgery and radiotherapy are current therapeutic options for malignant tumors involving the nasal vestibule. Depending on the location, organ-preserving resection is not always possible, even for small tumors. Definitive radiotherapy is an alternative as an organ-preserving procedure. Carbon ion beam radiotherapy offers highly conformal dose distributions and more complex biological radiation effects eventually resulting in optimized normal tissue sparing and improved outcome. The aim of the current study was to analyze toxicity, local control (LC), and organ preserving survival (OPS) after irradiation of carcinoma of the nasal vestibule with raster-scanned carbon ion radiotherapy boost (CIRT-B) combined with volumetric intensity modulated arc therapy (VMAT) with photons. METHODS: Between 12/2015 and 05/2021, 21 patients with malignant tumors involving the nasal vestibule were irradiated with CIRT-B combined with VMAT and retrospectively analyzed. Diagnosis was based on histologic findings. A total of 17 patients had squamous cell carcinoma (SCC) and 4 had other histologies. In this series, 10%, 67%, and 24% of patients had Wang stages 1, 2, and 3 tumors, respectively. Three patients had pathologic cervical nodes on MRI. The median CIRT-B dose was 24 Gy(RBE), while the median VMAT dose was 50 Gy. All patients with pathologic cervical nodes received simultaneously integrated boost with photons (SIB) up to a median dose of 62.5 Gy to the pathological lymph nodes. Eight patients received cisplatin chemotherapy. All patients received regular follow-up imaging after irradiation. Kaplan-Meier estimation was used for statistical assessment. RESULTS: The median follow-up after irradiation was 18.9 months. There were no common toxicity criteria grade 5 or 4 adverse events. A total of 20 patients showed grade 3 adverse events mainly on skin and mucosa. All patients were alive at the end of follow-up. The median OPS after treatment was 56.5 months. The 6- and 24-month OPS were 100% and 83.3%, respectively. All local recurrences occurred within 12 months after radiotherapy. The median progression free survival (PFS) after treatment was 52.4 months. The 6-, 12-, and 24-month PFS rates were 95%, 83.6%, and 74.3%, respectively. CONCLUSION: CIRT-B combined with VMAT in malignant tumors of the nasal vestibule is safe and feasible, results in high local control rates, and thus is a good option as organ-preserving therapy. No radiation-associated grade 4 or 5 acute or late AE was documented.

[The role of chemoradiotherapy in curative treatment of head and neck cancer]. / Kurative Radiochemotherapie von Kopf-Hals-Tumoren.

Radiotherapy and chemotherapy are integral parts of definitive and adjuvant therapy in the treatment of head and neck squamous cell carcinoma (HNSCC). The outcome of therapy is essentially dependent on selection and dosage of chemotherapeutical substances and on the other hand on the radiotherapeutical setting concerning fractionation, time of therapy and technical aspects. Immunotherapeutical substances have an increasing role in the therapy of HNSCC as well as particle therapy is investigated as part of radiotherapy in actual studies. Further challenges relate to treatment of HPV-induced tumors with regard to their differences in tumor biology and consecutively better prognosis.

Upfront Surgery vs. Primary Chemoradiation in an Unselected, Bicentric Patient Cohort with Oropharyngeal Squamous Cell Carcinoma-A Matched-Pair Analysis.

The two pillars of therapy for oropharyngeal squamous cell carcinoma (OPSCC) are upfront surgery and primary chemoradiotherapy. Substantial regional preferences exist with regard to the selection of treatment. Despite new therapeutic approaches, patient survival remains poor, with an approximate overall survival (OS) rate of 50% at five years. This study was conducted to investigate a potential survival benefit depending on the treatment modality in OPSCC patients. We retrospectively collected data of 853 patients with histologically confirmed OPSCC from the Giessen and Maastricht cancer databases. To identify risk factors affecting survival, a Cox-proportional hazard model was applied to 442 patients with complete data sets. Based on this cohort a matched-pair analysis with 158 patients was performed to compare OS rates of patients treated either with upfront surgery or primary chemoradiation. For the collective cohort, patients treated with upfront surgery had significantly improved OS rates compared to patients treated with primary chemoradiation. In the matched-pair analysis adjusted for patients' T-, N- and HPV-status as well as risk profile, we observed that both treatment approaches offered equivalent OS rates. Our study emphasizes that treatment recommendations should be made whenever possible on the basis of side-effect profiles caused by the therapeutic approach used. To draw further conclusions, results of the ongoing "best of" (NCT2984410) study are eagerly awaited, investigating the functional outcome after treatment of OPSCC patients.

Mutation patterns in recurrent and/or metastatic oropharyngeal squamous cell carcinomas in relation to human papillomavirus status.

Patients with HPV-driven (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) have a significantly improved overall survival compared to patients with HPV-negative (HPV-) OPSCC. Nevertheless, 13%-25% of patients with HPV+OPSCC develop local/distant recurrence (LDR) and have a course of disease similar to HPV-OPSCC. We hypothesize that HPV+OPSCCs of patients with LDR have a mutation frequency and pattern similar to HPV-OPSCCs, which is associated with severe outcome. We performed targeted next-generation sequencing using a customized gene panel and compared data from 56 matched HPV+and HPV-OPSCC of patients with/without LDR regarding protein-altering variants. Despite improved overall survival of patients with HPV+OPSCC, those who develop LDR show a strongly reduced survival rate that is similar or even worse compared to HPV-OPSCC patients. Overall, the number of mutations was similar in OPSCC of patients with and without LDR. In total and with respect to TP53, HPV-OPSCC had significantly more protein-altering mutations than HPV+OPSCC. The number of mutations was similar in HPV-OPSCC of patients with and without LDR with the exception of FAT1, which was mutated more frequently in patients without LDR. In HPV+OPSCC, HRAS, PIK3R1, STK11 and TP63 were more frequently mutated in patients with LDR compared to patients without. HPV+OPSCC of patients with LDR have a similar mutation pattern as HPV-OPSCC, except TP53, which was mutated to a significantly lower extent. In conclusion, HPV-and HPV+OPSCC with LDR have similar mutation counts in the analyzed genes. We suspect that the number of mutations is not causal for disease progression, rather specific mutations could be important.

Deep Learning Predicts HPV Association in Oropharyngeal Squamous Cell Carcinomas and Identifies Patients with a Favorable Prognosis Using Regular H&E Stains.

PURPOSE: Human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OPSCC) is tumorigenic and has been associated with a favorable prognosis compared with OPSCC caused by tobacco, alcohol, and other carcinogens. Meanwhile, machine learning has evolved as a powerful tool to predict molecular and cellular alterations of medical images of various sources. EXPERIMENTAL DESIGN: We generated a deep learning-based HPV prediction score (HPV-ps) on regular hematoxylin and eosin (H&E) stains and assessed its performance to predict HPV association using 273 patients from two different sites (OPSCC; Giessen, n = 163; Cologne, n = 110). Then, the prognostic relevance in a total of 594 patients (Giessen, Cologne, HNSCC TCGA) was evaluated. In addition, we investigated whether four board-certified pathologists could identify HPV association (n = 152) and compared the results to the classifier. RESULTS: Although pathologists were able to diagnose HPV association from H&E-stained slides (AUC = 0.74, median of four observers), the interrater reliability was minimal (Light Kappa = 0.37; P = 0.129), as compared with AUC = 0.8 using the HPV-ps within two independent cohorts (n = 273). The HPV-ps identified individuals with a favorable prognosis in a total of 594 patients from three cohorts (Giessen, OPSCC, HR = 0.55, P < 0.0001; Cologne, OPSCC, HR = 0.44, P = 0.0027; TCGA, non-OPSCC head and neck, HR = 0.69, P = 0.0073). Interestingly, the HPV-ps further stratified patients when combined with p16 status (Giessen, HR = 0.06, P < 0.0001; Cologne, HR = 0.3, P = 0.046). CONCLUSIONS: Detection of HPV association in OPSCC using deep learning with help of regular H&E stains may either be used as a single biomarker, or in combination with p16 status, to identify patients with OPSCC with a favorable prognosis, potentially outperforming combined HPV-DNA/p16 status as a biomarker for patient stratification.

Is Biofeedback through an Intra-Aural Device an Effective Method to Treat Bruxism? Case Series and Initial Experience.

Biofeedback was reported as an effective concept for bruxism treatment, through increasing patient's awareness of the habit. During bruxing both ear canals become tighter, therefore, an in-ear device can provide biofeedback. The in-ear device is fitted to the ear canal in physiological status, during bruxing the ear-canal tightens resulting in stress on the canal walls and unpleasant feeling. Subsequently, patients stop their bruxing habit. The aim of this study is to provide first clinical evidence that in-ear devices have a positive impact on relieving bruxism in patients. Despite the low number of patients, this early study was designed as a controlled prospective study. The trial included seven female patients with a median age of 47.3 years (23-64 years). Only two patients implemented their devices for eight and seven months, respectively. One patient reported a relief in her symptoms, like headaches and pain intensity during the night, by 50% after three month and 80% after six months. Despite the limited number of participants, the study reflects a potential of Intra-aural devices as effective biofeedback devices in treating bruxism.

Infectious KoRV-related retroviruses circulating in Australian bats.

Bats are reservoirs of emerging viruses that are highly pathogenic to other mammals, including humans. Despite the diversity and abundance of bat viruses, to date they have not been shown to harbor exogenous retroviruses. Here we report the discovery and characterization of a group of koala retrovirus-related (KoRV-related) gammaretroviruses in Australian and Asian bats. These include the Hervey pteropid gammaretrovirus (HPG), identified in the scat of the Australian black flying fox (Pteropus alecto), which is the first reproduction-competent retrovirus found in bats. HPG is a close relative of KoRV and the gibbon ape leukemia virus (GALV), with virion morphology and Mn2+-dependent virion-associated reverse transcriptase activity typical of a gammaretrovirus. In vitro, HPG is capable of infecting bat and human cells, but not mouse cells, and displays a similar pattern of cell tropism as KoRV-A and GALV. Population studies reveal the presence of HPG and KoRV-related sequences in several locations across northeast Australia, as well as serologic evidence for HPG in multiple pteropid bat species, while phylogenetic analysis places these bat viruses as the basal group within the KoRV-related retroviruses. Taken together, these results reveal bats to be important reservoirs of exogenous KoRV-related gammaretroviruses.

LAG-3, TIM-3 and VISTA Expression on Tumor-Infiltrating Lymphocytes in Oropharyngeal Squamous Cell Carcinoma-Potential Biomarkers for Targeted Therapy Concepts.

Tumor growth and survival requires a particularly effective immunosuppressant tumor microenvironment (TME) to escape destruction by the immune system. While immunosuppressive checkpoint markers like programmed cell death 1 ligand (PD-L1) are already being targeted in clinical practice, lymphocyte-activation-protein 3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA) inhibitors are currently under investigation in clinical trials. Reliable findings on the expression status of those immune checkpoint inhibitors on tumor-infiltrating lymphocytes (TILs) in the TME of oropharyngeal squamous cell carcinoma (OPSCC) are lacking. This work aims to describe the expression of LAG-3, TIM-3, and VISTA expression in the TME of OPSCC. We created a tissue microarray of paraffin-embedded tumor tissue of 241 OPSCC. Expression of the immune checkpoint protein LAG-3, TIM-3, and VISTA in OPSCC was evaluated using immunohistochemistry and results were correlated with CD8+ T-cell inflammation and human papillomavirus (HPV)-status. 73 OPSCC stained positive for LAG-3 (31%; HPV+:44%; HPV-:26%, p = 0.006), 122 OPSCC stained positive for TIM-3 (51%; HPV+:70%; HPV-:44%, p < 0.001) and 168 OPSCC (70%; HPV+:75%; HPV-:68%, p = 0.313) for VISTA. CD8+ T-cells were significantly associated with LAG-3, TIM-3 and VISTA expression (p < 0.001, p < 0.001, p = 0.007). Immune checkpoint therapy targeting LAG-3, TIM-3, and/or VISTA could be a promising treatment strategy especially in HPV-related OPSCC. Future clinical trials investigating the efficacy of a checkpoint blockade in consideration of LAG-3, TIM-3, and VISTA expression are required.

[Sialolithiasis: Current Diagnostics and Therapy]. / Sialolithiasis: aktuelle Diagnostik und Therapie.

Sialolithiasis is one of the most common salivary gland diseases. The main symptom is acute swelling of a major salivary gland after food intake, whereby the submandibular gland is significantly more frequently affected compared to the parotid gland. In the course of the disease, recurrent sialadenitis occurs in many cases. In addition to submandibulectomy, there is currently not only the possibility of improved diagnostics but also of gland-preserving therapy, for example by means of miniature endoscopy (sialendoscopy).

[Laryngeal Recurrent Respiratory Papillomatosis: Current Aspects on Diagnosis and Therapy]. / Rezidivierende respiratorische Papillomatose: Aktuelles zu Diagnose und Therapie.

Recurrent Respiratory Papillomatosis (RRP) is a rare infectious disease of the upper aerodigestive tract caused by human papillomavirus (HPV). Virus subtypes 6 and 11 are found in most of the papillomas, type 11 is associated with a severe course. There are a juvenile and an adult form defined by age of initial manifestation. The juvenile form shows a more severe course according to number of surgical interventions and secondary malignancy. A variety of treatment modalities are used to treat RRP. Baseline therapy is a surgical therapy via microlaryngoscopical approach. Extended surgical therapies are not indicated because HPV persists in surrounding tissue of visible lesions. Primary objective is therefore functional preservation of the aerodigestive tract. Adjuvant therapy might be helpful in selected cases, and is actually subject of investigation. HPV vaccination exists and will prospectively change incidence of RRP in the following years.

Factors influencing the induction of high affinity antibodies to Plasmodium falciparum merozoite antigens and how affinity changes over time.

Understanding the functional characteristics of naturally acquired antibodies against P. falciparum merozoite antigens is crucial for determining the protective functions of antibodies. Affinity (measured as kd) of naturally acquired antibodies against two key targets of acquired immunity, EBA175 and PfRh2, was determined using Surface Plasmon Resonance (SPR) in a longitudinal survey in Nigeria. A majority of the participants, 79% and 67%, maintained stable antibody affinities to EBA175 and PfRh2, respectively, over time. In about 10% of the individuals, there was a reciprocal interaction with a reduction over time in antibody affinity for PfRh2 and an increase for EBA175. In general, PfRh2 elicited antibodies with higher affinity compared to EBA175. Individuals with higher exposure to malaria produced antibodies with higher affinity to both antigens. Younger individuals (5-15 years) produced comparable or higher affinity antibodies than adults (>15 years) against EBA175, but not for PfRh2. Correlation between total IgG (ELISA) and affinity varied between individuals, but PfRh2 elicited antibodies with a higher correlation in a majority of the participants. There was also a correlation between antibody inhibition of erythrocyte invasion by merozoites and PfRh2 affinity. This work gives new insights into the generation and maintenance of antibody affinity over time.

Identifying Immune Correlates of Protection Against Plasmodium falciparum Through a Novel Approach to Account for Heterogeneity in Malaria Exposure.

Background: A main criterion to identify malaria vaccine candidates is the proof that acquired immunity against them is associated with protection from disease. The age of the studied individuals, heterogeneous malaria exposure, and assumption of the maintenance of a baseline immune response can confound these associations. Methods: Immunoglobulin G/immunoglobulin M (IgG/ IgM) levels were measured by Luminex® in Mozambican children monitored for clinical malaria from birth until 3 years of age, together with functional antibodies. Studied candidates were pre-erythrocytic and erythrocytic antigens, including EBAs/PfRhs, MSPs, DBLs, and novel antigens merely or not previously studied in malaria-exposed populations. Cox regression models were estimated at 9 and 24 months of age, accounting for heterogeneous malaria exposure or limiting follow-up according to the antibody's decay. Results: Associations of antibody responses with higher clinical malaria risk were avoided when accounting for heterogeneous malaria exposure or when limiting the follow-up time in the analyses. Associations with reduced risk of clinical malaria were found only at 24 months old, but not younger children, for IgG breadth and levels of IgG targeting EBA140III-V, CyRPA, DBL5ε and DBL3x, together with C1q-fixation activity by antibodies targeting MSP119. Conclusions: Malaria protection correlates were identified, only in children aged 24 months old when accounting for heterogeneous malaria exposure. These results highlight the relevance of considering age and malaria exposure, as well as the importance of not assuming the maintenance of a baseline immune response throughout the follow-up. Results may be misleading if these factors are not considered.