Fetal Zika virus inoculation in macaques revealed control of the fetal viral load during pregnancy.

BACKGROUND: Early pregnancy Zika virus (ZIKV) infection is associated with major brain damage in fetuses, leading to microcephaly in 0.6-5.0% of cases, but the underlying mechanisms remain largely unknown. METHODS: To understand the kinetics of ZIKV infection during fetal development in a nonhuman primate model, four cynomolgus macaque fetuses were exposed in utero through echo-guided intramuscular inoculation with 103 PFU of ZIKV at 70-80 days of gestation, 2 controls were mock inoculated. Clinical, immuno-virological and ultrasound imaging follow-ups of the mother/fetus pairs were performed until autopsy after cesarean section 1 or 2 months after exposure (n = 3 per group). RESULTS: ZIKV was transmitted from the fetus to the mother and then replicate in the peripheral blood of the mother from week 1 to 4 postexposure. Infected fetal brains tended to be smaller than those of controls, but not the femur lengths. High level of viral RNA ws found after the first month in brain tissues and placenta. Thereafter, there was partial control of the virus in the fetus, resulting in a decreased number of infected tissue sections and a decreased viral load. Immune cellular and humoral responses were effectively induced. CONCLUSIONS: ZIKV infection during the second trimester of gestation induces short-term brain injury, and although viral genomes persist in tissues, most of the virus is cleared before delivery.

Assessment of blood viral load in asymptomatic and symptomatic cases of congenital cytomegalovirus infection.

This study aimed to retrospectively evaluate the baseline and follow-up viral loads and viral clearance times in cases followed for asymptomatic and symptomatic congenital cytomegalovirus (cCMV) infection between August 2010 and August 2022. Among 93 cases, they had asymptomatic (n: 55) and symptomatic (n: 38). The median baseline blood viral load detected in the symptomatic cCMV (ScCMV) infection (13 054 IU/mL) was significantly higher than that of asymptomatic cCMV (AcCMV) infection (4636 IU/mL) (p < 0.013). There was no difference in median viral clearance times (75 and 90 days, respectively) in baseline viremic cases in the ScCMV and AcCMV infection groups. There were no differences in median baseline blood viral load (6930 IU/mL and 14 268 IU/mL, respectively) and median viral clearance times (75 and 85 days, respectively) between the 6-week and 6-month antiviral treatment group. No correlation was found between baseline blood viral load, clinical severity, and the number of systems involved. However, in initial viremic cases, the viral load threshold for a symptomatic case was 8856 IU/mL, with 85.7% sensitivity and 54.5% specificity.

Mechanistic modeling of minute virus of mice surrogate removal by anion exchange chromatography in micro scale.

Biopharmaceutical products are often produced in Chinese hamster ovary (CHO) cell cultures that are vulnerable to virus infections. Therefore, it is a regulatory requirement that downstream purification steps for biopharmaceuticals can remove viruses from feedstocks. Anion exchange chromatography (AEX) is one of the downstream unit operations that is most frequently used for this purpose and claimed for its capability to remove viruses. However, the impact of various process parameters on virus removal by AEX is still not fully understood. Mechanistic modeling could be a promising way to approach this gap, as these models require comparatively few experiments for calibration. This makes them a valuable tool to improve understanding of viral clearance, especially since virus spiking studies are costly and time consuming. In this study, we present how the virus clearance of a MVM mock virus particle by Q Sepharose FF resin can be described by mechanistic modeling. A lumped kinetic model was combined with a steric mass action model and calibrated at micro scale using three linear gradient experiments and an incremental step elution gradient. The model was subsequently verified for its capability to predict the effect of different sodium chloride concentrations, as well as residence times, on virus clearance and was in good agreement with the LRVs of the verification runs. Overall, models like this could enhance the mechanistic understanding of viral clearance mechanisms and thereby contribute to the development of more efficient and safer biopharmaceutical downstream processes.

Risk management for viral clearance: A case study on adoption of platform validation approach and risk management of process changes.

Viral clearance (VC) studies are routinely required prior to entering clinical trials or for commercial launch of biopharmaceuticals. With increasing prior knowledge and experience, platform validation can be used to eliminate some VC studies and such strategy has been updated into industry guidelines, such as ICH Q5A (R2). In addition, process changes can happen during life-cycle management of a product. In these circumstances, high-risk process parameters need to be identified and corresponding control strategies need to be defined to ensure viral safety of the product. This work describes the design of a science-based risk management tool and how this tool is employed for platform validation and process change scenarios.

Infectivity and pathogenesis characterization of getah virus (GETV) strain via different inoculation routes in mice.

In recent years, the epidemiological profile of Getah virus (GETV) has become increasingly serious, posing a huge threat to animal and public health in China. GETV can cause multi-species infection, including horses, pigs, rats, cattle, kangaroos, reptiles and birds. However, there were few reports on the efficiency of the virus entering the host via routes of different systems. In the present study, a GETV strain (SC201807) was obtained from a piglet's blood in 2018 in Sichuan, China. First, we established a quantitative real-time polymerase chain reaction (qRT-PCR) SYBR assay specific to GETV. Then, we evaluated the infection efficiency of different routes using mouse animal model. 108 male mice were randomly divided into four groups as follows: intramuscular, intraoral and intranasal infection routes, and negative control. All mice in the experimental group were inoculated with 4 × 102.85 TCID50 GETV virus. Tissue tropism experiments show that GETV has a wide range of tissue distribution, and intramuscular infection is the first to infect all tissues of the body, and suggest that oral infection may be a new GETV transmission route. Histopathological examination results showed that intramuscular injection of GETV mainly caused different degrees of pathological damage to the tissues, and could rapidly induce a large amount of inflammatory regulatory factors such as IL-6 and TNF-α. Our data may help us to evaluate the risk of transmission of Porcine Getah virus and provide an experimental basis for the prevention and control of Porcine Getah virus.

Exploring early COVID-19 therapies, variants, and viral clearance dynamics: Insights from a high-risk outpatients study.

This retrospective observational study investigates the impact of early COVID-19 therapies, including antivirals and monoclonal antibodies (mAbs), on time to achieve negative swab results in high-risk outpatients infected with specific Omicron sublineages. The study enrolled 104 patients from Luigi Sacco Hospital in Milan between December 2021 and March 2023, categorizing them based on the Omicron sublineage they were infected with (BA.1, BA.2, BA.4/BA.5) and the early treatment they received (antivirals or mAbs). Key data collected included demographic and clinical characteristics, initial and follow-up cycle threshold (Ct) values from qPCR tests, and the interval between swabs. The median age of the participants was 63 years (Interquartile Range [IQR] 54.0-76.5), and 55.8% were male. Among the patients, 15 received mAbs (14.4%), and 99 received antiviral treatments (95.2%) - specifically, Paxlovid (51.9%), Molnupiravir (21.1%), and Remdesivir (12.5%). No patients required hospitalization or experienced mortality during the one-month follow-up period. Regarding Omicron sublineages, 23 patients (22.1%) were infected with BA.1, 53 (51%) with BA.2, and 28 (26.9%) with BA.4/BA.5. The median interval between the initial and follow-up swabs was 6 days (IQR 6.0-7.0). Initial Ct values had a median of 18.5 (IQR 16.5-22.1), which increased to a median of 30.5 (IQR 27.1-33.0) at follow-up, indicating a reduction in viral load. A non-significant trend suggested that patients infected with BA.2 and BA.4/BA.5 sublineages might experience a faster increase in Ct values-indicating quicker viral load reduction - compared to those infected with BA.1, regardless of treatment type. However, this trend did not achieve statistical significance (p=0.609), likely due to the limited sample size and the absence of a clear trend curve. In summary, the study did not find a significant association between specific early therapies and the time to achieve swab negativization. These findings underscore the complex dynamics of viral clearance and highlight the need for further research with larger patient cohorts to refine treatment protocols for high-risk COVID-19 patients.

Relationship between acute SARS-CoV-2 viral clearance with Long COVID Symptoms: a cohort study.

Introduction: The relationship between SARS-CoV-2 viral dynamics during acute infection and the development of long COVID is largely unknown. Methods: A total of 7361 asymptomatic community-dwelling people enrolled in the Test Us at Home parent study between October 2021 and February 2022. Participants self-collected anterior nasal swabs for SARS-CoV-2 RT-PCR testing every 24-48 hours for 10-14 days, regardless of symptom or infection status. Participants who had no history of COVID-19 at enrollment and who were subsequently found to have ≥1 positive SARS-CoV-2 RT-PCR test during the parent study were recontacted in August 2023 and asked whether they had experienced long COVID, defined as the development of new symptoms lasting 3 months or longer following SARS-CoV-2 infection. Participant's cycle threshold values were converted into viral loads, and slopes of viral clearance were modeled using post-nadir viral loads. Using a log binomial model with the modeled slopes as the exposure, we calculated the relative risk of subsequently developing long COVID with 1-2 symptoms, 3-4 symptoms, or 5+ symptoms, adjusting for age, number of symptoms, and SARS-CoV-2 variant. Adjusted relative risk (aRR) of individual long COVID symptoms based on viral clearance was also calculated. Results: 172 participants were eligible for analyses, and 59 (34.3%) reported experiencing long COVID. The risk of long COVID with 3-4 symptoms and 5+ symptoms increased by 2.44 times (aRR: 2.44; 95% CI: 0.88-6.82) and 4.97 times (aRR: 4.97; 95% CI: 1.90-13.0) per viral load slope-unit increase, respectively. Participants who developed long COVID had significantly longer times from peak viral load to viral clearance during acute disease than those who never developed long COVID (8.65 [95% CI: 8.28-9.01] vs. 10.0 [95% CI: 9.25-10.8]). The slope of viral clearance was significantly positively associated with long COVID symptoms of fatigue (aRR: 2.86; 95% CI: 1.22-6.69), brain fog (aRR: 4.94; 95% CI: 2.21-11.0), shortness of breath (aRR: 5.05; 95% CI: 1.24-20.6), and gastrointestinal symptoms (aRR: 5.46; 95% CI: 1.54-19.3). Discussion: We observed that longer time from peak viral load to viral RNA clearance during acute COVID-19 was associated with an increased risk of developing long COVID. Further, slower clearance rates were associated with greater number of symptoms of long COVID. These findings suggest that early viral-host dynamics are mechanistically important in the subsequent development of long COVID.

A Robust SARS-CoV-2-specific T and B Cell Response is Associated with Early Viral Clearance in SARS-CoV-2 Omicron-Infected Immunocompromised Individuals.

BACKGROUND: The immunological determinants of delayed viral clearance and intra-host viral evolution that drive the development of new pathogenic virus strains in immunocompromised individuals are unknown. Therefore, we longitudinally studied SARS-CoV-2-specific immune responses in relation to viral-clearance and evolution in immunocompromised individuals. METHODS: Among Omicron-infected immunocompromised individuals, we determined SARS-CoV-2-specific T- and B-cell responses, anti-spike IgG(3) titers, neutralization titers, and monoclonal antibody (mAb)-resistance-associated mutations. The 28-day post-enrollment nasopharyngeal specimen defined early (RT-PCR negative ≤28 days) or late (RT-PCR- positive >28 days) viral-clearance. RESULTS: Of 30 patients included (median age 61.9 years [IQR 47.4-72.3], 50% females), 20 (66.7%) received mAb-therapy. Thirteen (43.3%) demonstrated early and 17 (56.7%) late viral-clearance. Early viral-clearance patients and patients without resistance-associated mutations had significantly higher baseline IFN-γ release and early viral-clearance patients had a higher frequency of SARS-CoV-2-specific B-cells at baseline. In non-mAb-treated patients, day 7 IgG and neutralization titers were significantly higher in those with early versus late viral-clearance. CONCLUSION: An early robust adaptive immune response is vital for efficient viral-clearance and associated with less emergence of mAb-resistance-associated mutations in Omicron-infected immunocompromised patients. This emphasizes the importance of early SARS-CoV-2-specific T- and B-cell responses and thereby provides a rationale for development of novel therapeutic approaches.

Differential Gene Expression in the Upper Respiratory Tract following Acute COVID-19 Infection in Ambulatory Patients That Develop Long COVID.

Background: Post-acute sequelae of COVID-19, or long COVID, is a condition characterized by persistent COVID-19 symptoms. As long COVID is defined by clinical criteria after an elapsed period, an opportunity for early intervention may aid in future prophylactic approaches; however, at present, the pathobiological mechanisms are multifactorial. By analyzing early virally infected upper respiratory tract tissue prior to eventual clinical diagnosis, it may be possible to identify biomarkers of altered immune response to facilitate future studies and interventions. Methods: This is a sub-group analysis of samples collected from those with confirmed COVID-19. RNA extraction from nasopharyngeal/mid-turbinate samples, sequencing, and bioinformatic analysis were performed to analyze long COVID and non-long COVID cohorts at day 14 post infection. Differences in mean viral load at various timepoints were analyzed as well as serological data. Results: We identified 26 upregulated genes in patients experiencing long COVID. Dysregulated pathways including complement and fibrinolysis pathways and IL-7 upregulation. Additionally, genes involved in neurotransmission were dysregulated, and the long COVID group had a significantly higher viral load and slower viral clearance. Conclusions: Uncovering early gene pathway abnormalities associated with eventual long COVID diagnosis may aid in early identification. We show that, post acute infection, in situ pathogenic deviations in viral response are associated with patients destined to meet consensus long COVID diagnosis that is entirely dependent on clinical factors. These results identify an important biological temporal window in the natural history of COVID-19 infection and long COVID pathogenesis amenable to testing from standard-of-care upper respiratory tract specimens.

Real-world effectiveness of molnupiravir, azvudine and paxlovid against mortality and viral clearance among hospitalized patients with COVID-19 infection during the omicron wave in China: A retrospective cohort study.

OBJECTIVES: In this retrospective cohort study, we aimed to assess clinical effectiveness and viral clearance following the use of molnupiravir, azvudine and paxlovid in hospitalized patients with COVID-19 in China dominated by the omicron BA.5.2 and BF.7 subvariant of SARS-CoV-2. METHODS: Enrolled patients were assigned to the molnupiravir group or the azvudine group or the paxlovid group or the control group (not taking any antiviral drugs). The primary outcome of the cohort study was viral clearance and viral burden rebound after treatment and the secondary outcome was 28-day all-cause mortality. The four groups were propensity score-matched (1:1). We plotted viral load trends for each antiviral drug intervention using locally weighted regression (LOWESS) smoothed data. Multivariate logistic regression (stepwise algorithm) models were used to determine any risk factors for 28-day mortality. RESULTS: Of the 1537 patients receiving any treatment, 886 (57.6 %) received molnupiravir, 390 (25.4 %) received azvudine, 94 (6.1 %) received paxlovid, and 167 (10.9 %) did not use any antiviral drugs. Our data analysis showed that age (OR = 1.05, 95 % CI: 1.03-1.07, P < 0.001), Charlson comorbidty index (OR = 1.32, 95 % CI: 1.18-1.48, P < 0.001), severity of COVID-19 (P < 0.001), gamma globulin (OR = 2.04, 95 % CI: 1.03-3.99, P = 0.039) and corticosteroids use (OR = 2.3, 95 % CI: 1.19-4.69, P = 0.017) were independent prognostic factors for 28-day mortality in COVID-19 patients. After propensity score matching (PSM), the paxlovid recipients (OR = 0.22, 95 % CI: 0.05-0.83, P = 0.036) or azvudine recipients (OR = 0.27, 95 % CI: 0.07-0.91, P = 0.046) had lower 28-day mortality compared to their matched controls. Viral rebound occurred in the control group around days 9-16, while no viral rebound was found in any of the three oral antiviral groups. We found that molnupiravir group performed comparably in terms of the rate of nucleic acid conversion negative compared with the paxlovid group, while azvudine group performed slightly worse compared with the paxlovid group or molnupiravir group. CONCLUSIONS: In our retrospective cohort of hospitalized patients with COVID-19 during the wave of omicron strain, the molnupiravir, paxlovid and azvudine recipients showed a faster and more stable decrease in viral load and rare virus rebound in response to antiviral treatments when compared to the controls. The study supported that initiation treatment with paxlovid and azvudine was associated with significantly lower risk of all-cause death within 28 days.

Comparison of time to negative conversion of SARS-CoV-2 between young and elderly among asymptomatic and mild COVID-19 patients: a cohort study from a national containment center.

Objective: We aimed to study the relationship between age and time to negative conversion of SARS-CoV-2 in patients with asymptomatic and mild forms of COVID-19. Methods: We conducted a cohort study including all patients diagnosed with COVID-19 from the national COVID-19 containment center of Tunisia. Patients were subdivided into two cohorts: (under 60 years) and (over 60 years) and were followed up until PCR negativization. Log rank test and Cox regression were applied to compare time to negative conversion between the old group and the young group. Results: The study included 289 patients with non-severe forms of COVID-19. Age over 60 was significantly associated with delayed negative conversion in male sex (Hazard ratio (HR): 1.9; 95% CI: 1.2-3.07) and among patients with morbid conditions (HR:1.68; 95% CI: 1.02-2.75) especially diabetics (HR: 2.06; 95% CI: 1.01-4.21). This association increased to (HR:2.3; 95% CI: 1.13-4.66) when male sex and comorbidities were concomitantly present and rose to (HR: 2.63; 95% CI: 1.02-6.80) for men with diabetes. Cox regression analysis revealed a significantly delayed negative conversion in symptomatic patients. Significant interaction was observed between gender and age and between age and chronic conditions. Conclusion: Age is associated with delayed negative conversion of viral RNA in certain subgroups. Identifying these subgroups is crucial to know how prioritize preventive strategies in elderly.

The role of long-lived plasma cells in viral clearance.

The adaptive immune system has two types of plasma cells (PC), long-lived plasma cells (LLPC) and short-lived plasma cells (SLPC), that differ in their lifespan. In this paper, we propose that LLPC is crucial to the clearance of viral particles in addition to reducing the viral basic reproduction number in secondary infections. We use a sequence of within-host mathematical models to show that, CD8 T cells, SLPC and memory B cells cannot achieve full viral clearance, and the viral load will reach a low positive equilibrium level because of a continuous replenishment of target cells. However, the presence of LLPC is crucial for viral clearance.

CHO cells for virus-like particle and subunit vaccine manufacturing.

Chinese Hamster Ovary (CHO) cells, employed primarily for manufacturing monoclonal antibodies and other recombinant protein (r-protein) therapeutics, are emerging as a promising host for vaccine antigen production. This is exemplified by the recently approved CHO cell-derived subunit vaccines (SUV) against respiratory syncytial virus (RSV) and varicella-zoster virus (VZV), as well as the enveloped virus-like particle (eVLP) vaccine against hepatitis B virus (HBV). Here, we summarize the design, production, and immunogenicity features of these vaccine and review the most recent progress of other CHO-derived vaccines in pre-clinical and clinical development. We also discuss the challenges associated with vaccine production in CHO cells, with a focus on ensuring viral clearance for eVLP products.

Viral clearance capability of monoclonal antibody purification.

Viral clearance steps are routinely included in monoclonal antibody purification processes to safeguard product from potential virus contamination. These steps are often experimentally studied using product-specific feeds and parameters for each project to demonstrate viral clearance capability. However, published evidence suggests that viral clearance capability of many of these steps are not significantly impacted by variations in feed material or process parameter within commonly used ranges. The current investigation confirms robust retrovirus inactivation by low pH treatment and parvovirus removal by second-generation virus filters, independent to individual antibody molecules. Our results also reveal robust retrovirus removal by flowthrough anion exchange chromatography, inside the limits of protein load and host cell protein content. The cumulative viral clearance capability from these steps leads to an excess clearance safety factor of 10,000-fold for endogenous retrovirus-like particles. These results further justify the use of prior knowledge-based modular viral clearance estimation as opposed to repetitive experimentation.

Viral inactivation for pH-sensitive antibody formats such as multi-specific antibodies.

Recently developed multi-specific antibody formats enable new therapeutic concepts. Conveniently, formats with an Fc domain allow purification in well-established mAb platform processes. However, due to the structural complexity of the formats, the assembled molecules may be sensitive to extreme pH commonly used for viral inactivation. An alternative to low pH incubation for virus inactivation is the use of a mixture of tri-n-butyl phosphate (TnBP, solvent) and Polysorbate 80 (PS80, detergent). While TnBP is toxic, this combination has a long history of use in the manufacturing of human plasma-derived products that are sensitive to low or high pH incubation. Data are provided demonstrating that the solvent/detergent (S/D) treatment using TnBP and PS80 can be successfully used for pH-sensitive, multi-specific antibody formats in the clarified cell culture fluid (CCCF). A different placement of the S/D within the purification process, namely during the capture by Protein A (PA), has been evaluated. This alternative placement allows effective viral inactivation by S/D while preserving the viral reduction and viral inactivation achieved through the PA step itself, enabling the cumulation of these effects. Furthermore, the process alternative simplifies the liquid handling by reducing the added volumes of the required S/D liquids, thus reducing the amount of toxic TnBP to a minimum. Data are shown demonstrating a complete removal of TnBP and PS80 in the process.

Efficient formation and maintenance of humoral and CD4 T-cell immunity targeting the viral capsid in acute-resolving hepatitis E infection.

BACKGROUND & AIMS: CD4 T cells shape the neutralizing antibody (nAb) response and facilitate viral clearance in various infections. Knowledge of their phenotype, specificity and dynamics in hepatitis E virus (HEV) infection is limited. HEV is enterically transmitted as a naked virus (nHEV) but acquires a host-derived quasi-envelope (eHEV) when budding from cells. While nHEV is composed of the open reading frame (ORF)-2-derived capsid, eHEV particles also contain ORF3-derived proteins. We aimed to longitudinally characterize the HEV-specific CD4 T cells targeting ORF1, 2 and 3 and antibodies against nHEV or eHEV in immunocompetent individuals with acute and resolved HEV infection. METHODS: HEV-specific CD4 T cells were analyzed by intracellular cytokine staining after stimulation with in silico-predicted ORF1- and ORF2-derived epitopes and overlapping peptides spanning the ORF3 region. Ex vivo multiparametric characterization of capsid-specific CD4 T cells was performed using customized MHC class II tetramers. Total and neutralizing antibodies targeting nHEV or eHEV particles were determined. RESULTS: HEV-specific CD4 T-cell frequencies and antibody titers are highest in individuals with acute infection and decline in a time-dependent process with an antigen hierarchy. HEV-specific CD4 T cells strongly target the ORF2-derived capsid and ORF3-specific CD4 T cells are hardly detectable. NAbs targeting nHEV are found in high titers while eHEV particles are less efficiently neutralized. Capsid-specific CD4 T cells undergo memory formation and stepwise contraction, accompanied by dynamic phenotypical and transcriptional changes over time. CONCLUSION: The viral capsid is the main target of HEV-specific CD4 T cells and antibodies in acute-resolving infection, correlating with efficient neutralization of nHEV. Capsid-specific immunity rapidly emerges followed by a stepwise contraction several years after infection. IMPACT AND IMPLICATIONS: The interplay of CD4 T cells and neutralizing antibody responses is critical in the host defense against viral infections, yet little is known about their characteristics in hepatitis E virus (HEV) infection. We conducted a longitudinal study of immunocompetent individuals with acute and resolved HEV infection to understand the characteristics of HEV-specific CD4 T cells and neutralizing antibodies targeting different viral proteins and particles. We found that HEV-specific CD4 T cells mainly target capsid-derived epitopes. This correlates with efficient neutralization of naked virions while quasi-enveloped particles are less susceptible to neutralization. As individuals with pre-existing liver disease and immunocompromised individuals are at risk for fulminant or chronic courses of HEV infection, these individuals might benefit from the development of vaccination strategies which require a detailed knowledge of the composition and longevity of HEV-specific CD4 T-cell and antibody immunity.

Adapting virus filtration to continuous processing: Effects of product and process variability on filtration performance.

Virus filtration (VF) is an important unit operation in the manufacture of biotherapeutics that provides robust removal of potential virus contaminants. Small virus removal can be impacted by the low operating pressures and potential depressurization events that are often associated with continuous operations where increased operational flexibility for higher loading at low flux and low pressure is required. In this study, we evaluated the impact of low flux (7 LMH) and pressure interruptions on minute virus of mice (MVM) removal. We used long-term filtrations conducted to a target throughput of 1000 L/m2 with four different monoclonal antibodies on small-scale hollow fiber virus filters with a hydrophilic modified polyvinylidene fluoride membrane. These conditions are certainly challenging for any VF operation and ensuring robust viral clearance under such conditions is critical to the design and implementation of continuous VF. Planova BioEX filters effectively removed MVM at 4 log or greater when run continuously for up to 6 days. Interestingly, pressure increases associated with filter fouling over the duration of long-term filtrations were shown to be reflective of load material variability and could be remediated by implementation of an inline prefilter. Pressure interruptions had minimal impact on overall MVM logarithmic reduction value. Effective virus removal was achieved with pressure increases being largely product-specific, which demonstrates the capability of the virus filter to remove virus independent of pressure increases that are expected to occur with increased protein load.

Immune Responses in Oral Papillomavirus Clearance in the MmuPV1 Mouse Model.

Human papillomavirus (HPV)-induced oropharyngeal cancer now exceeds HPV-induced cervical cancer, with a noticeable sex bias. Although it is well established that women have a more proficient immune system, it remains unclear whether immune control of oral papillomavirus infections differs between sexes. In the current study, we use genetically modified mice to target CCR2 and Stat1 pathways, with the aim of investigating the role of both innate and adaptive immune responses in clearing oral papillomavirus, using our established papillomavirus (MmuPV1) infection model. Persistent oral MmuPV1 infection was detected in Rag1ko mice with T and B cell deficiencies. Meanwhile, other tested mice were susceptible to MmuPV1 infections but were able to clear the virus. We found sex differences in key myeloid cells, including macrophages, neutrophils, and dendritic cells in the infected tongues of wild type and Stat1ko mice but these differences were not observed in CCR2ko mice. Intriguingly, we also observed a sex difference in anti-MmuPV1 E4 antibody levels, especially for two IgG isotypes: IgG2b and IgG3. However, we found comparable numbers of interferon-gamma-producing CD8 T cells stimulated by E6 and E7 in both sexes. These findings suggest that males and females may use different components of innate and adaptive immune responses to control papillomavirus infections in the MmuPV1 mouse model. The observed sex difference in immune responses, especially in myeloid cells including dendritic cell (DC) subsets, may have potential diagnostic and prognostic values for HPV-associated oropharyngeal cancer.

Evaluating the viral clearance ability of continuous monoclonal antibody purification steps, in order to inactivate and/or remove four model viruses.

Background and Objectives: Viral clearance studies are an essential part of a manufacturer's plan to ensure the safety of an injectable biologic product. In this way, viral safety is a critical quality attribute for biologics such as monoclonal antibodies (Mabs). Evaluation of virus purification by downstream processes is a key component of risk mitigation. In this study, the capability of continuous monoclonal antibody purification steps was evaluated in the process of instant monoclonal antibody purification in different stages of purification, and the amount of reduction or inactivation of each step was determined. Materials and Methods: Four enveloped and non-enveloped viral models VSV, Reovirus, EMCV, and HSV1 were used for spiking in selected samples in the designated tests, to have a comprehensive examination of the ability to clear the virus such as the type of genetic material, chemical resistance, and particle size. A TCID50 and qPCR methods were used to measure viral reduction. Two cell lines, Vero (African green monkey kidney) and L929 (Mouse fibroblast) were used for 4 model viruses propagation. The steps that were evaluated included 4 steps monoclonal antibody purification; cation exchange chromatography, acidic pH treatment, affinity chromatography, and nanofiltration. Results: The nano-filter stage showed the highest viral reduction and cation exchange chromatography showed the lowest reduction. The cumulative decrease using TCID50 is equal to 19.27 [log10] for all steps and for the qPCR method is equal to 12.47 [log10] in three steps of nano-filter, affinity chromatography, and ion exchange chromatography. Conclusion: The overall average reduction coefficient for all four model viruses is significantly high, which indicates the high capacity of the monoclonal antibody production process in inactivating and removing viruses leads to reducing the load of all four model viruses.