Neutralizing Activity Research Articles

Abstract 1368: Novel mRNA encoding anti-PD-L1 monoclonal antibodies for cancer immunotherapy

Abstract The difficulties with antibody-based therapies stem from their steep costs, complex procedures, and contamination risks. We investigate the use of in vitro transcription (IVT) for mRNA production, presenting a novel approach to traditional antibody treatments by promoting internal protein or antibody synthesis. We aim to create innovative mRNA-based anti-PD-L1 antibodies with strong immunotherapeutic effectiveness. The study begins with mouse immunization and hybridoma generation. Immunizing mice with the glycosylated extracellular domain of PD-L1 produces mouse antibodies against human PD-L1. Hybridomas, derived from post-immunization splenocytes, yield monoclonal antibodies screened for PD-L1 binding affinity via flow cytometry. A neutralizing assay identifies 22 antibodies with similar IC50 values and shared binding epitopes. M1, chosen for glycospecificity, affinity, and neutralizing activity, undergoes additional evaluation for the expression and anti-tumor efficacy of mRNA-encoded antibodies. The expression of M1 and atezolizumab antibodies is facilitated through an mRNA-lipid nanoparticles (LNPs) delivery system. Following intravenous administration in mice, sustained serum levels of M1 (ranging from 100 to 700 μg/mL at 24 hours post-injection) are observed. Serum concentrations of M1 and atezolizumab antibody peak at the first timepoint (24 or 48 h) after mRNA-LNP injection, respectively, and then significantly decrease within the next 4 days, with a gradual decline over the next 2 weeks. Conversely, the serum concentrations of M1 or atezolizumab protein decline significantly 24 hours post-injection and consistently stay at a baseline level (t1/2 of 94 and 4 h for M1 mRNA-LNP and protein, respectively). This indicates that the injection of encoded mRNA in mice can stably and continuously express antibodies.Administering Atezolizumab mRNA-LNP through two intravenous injections leads to a significant decrease in MC38 tumor growth, exhibiting a dose-dependent effect (TGI% = 8.1, 43.1, and 62.8 for doses of 0.2, 0.6, and 1 mg/kg, respectively). A complete tumor regression was observed up to day 19 following injection with a 2 mg/kg dose of mRNA-LNP (TGI% = 90.8%). Remarkably, a 3 mg/kg dose of atezolizumab antibody demonstrates a similar reduction in tumor growth as 0.6 mg/kg of the mRNA-LNP formulation in this model. In conclusion, this research underscores the potential of LNP-formulated mRNA as a transformative approach, converting the human body into a site for the continuous, stable expression of antibodies, offering a promising alternative to traditional protein-based therapies for combating cancer. This innovative approach shows promise in overcoming the limitations associated with traditional antibody production and clinical utilization. Citation Format: Ezra Chung, Yong Sik Bong, Renxiang Chen, Michael Zhang, Steven Long, Dong Shen. Novel mRNA encoding anti-PD-L1 monoclonal antibodies for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1368.

Abstract 4185: Neutralizing human IFN-β combined with anti-human PD-L1 and anti-human CTLA-4 treatment increased control of endometrial tumor growth in a novel HLA-A matched BLT humanized mice

Abstract High concentrations of type I interferon can induce growth arrest and apoptosis in cancer cells upon acute exposure, while low concentrations of the same can provide significant pro-survival benefits upon chronic exposure. In vitro data show the dependence of cancer cells on self-generated IFN-I leads to their demise if the IFN-β gene is disrupted, but they manage to persist when provided with IFN-β from an external source. Importantly, murine models show that inhibition of myeloid-associated INF-β can limit tumor growth. As blockage of type I interferon can also restore the CD8+ T cell function after chronic viral infection, we sought to determine the effects of neutralizing anti-human IFN-β antibody 7859 (N-anti-IFN-β) when added to T-cell directed immunotherapy in BLT humanized mice implanted with HLA-A matched human endometrial cancer PDX specimens. Specifically, we tested the combination of immunotherapy with N-anti-IFN-β combined with anti-human PD-L1 and anti-human CTLA-4. N-anti-IFN-β was first characterized for neutralizing activity to confirm specific neutralization of IFNβ and not human IFN-β. HLA-A matched genotyped CD34 stem cells and thymus (BLT humanized mice) and clinical endometrial tumors (PDX) were joined in BLT humanized mice before infusions with N-anti-IFN-β with or without anti-human PD-L1 and anti-human CTLA-4, anti-human PD-L1 and anti-human CTLA-4 alone, and IgG control antibodies intraperitoneally twice per week over three weeks. Tumor sizes were measured weekly and weighted at the end of the study. Human leukocytes infiltrating PDX were assessed by IHC and flow cytometry. Leukocyte subset activation, exhaustion, and tumor-specific T-cell function were measured. Anti-human PD-L1 and anti-human CTLA-4 reduced tumor growth by 82.9% (p<0.0001), but quickly increased the T cell exhaustion markers PD-1 and LAG3. The addition of N-anti-IFN-β to immunotherapy significantly decreased the tumor growth rate by 96.2% (p<0.0001) also significantly different from immunotherapy alone (p=0.0009) N-anti-IFN-β monotherapy reduced tumor growth rate by 42.4% (p=0.0032). While there was an increase in infiltrating CD4 and CD8 T-cells in combination therapy, reductions in expression of PD-1 and LAG3 were observed when compared to immunotherapy or N-IFN-β groups alone Analysis was done with Prism software. The use of a novel HLA-A matched BLT humanized mice with human tumor PDX model shows that neutralization of IFN-β can increase the T-cell anti-tumor activity and tumor repression of immunotherapy with anti-human PD-L1 and anti-human CTLA-4 treatment. Citation Format: Zhe Yuan, Guorui Zu, Xiao Sun, Emmanouil Papasavvas, Lily Lu, Joel Cassel, Peter L. Lutes, Mark Cadungog, Joseph Salvino, Luis J. Montaner. Neutralizing human IFN-β combined with anti-human PD-L1 and anti-human CTLA-4 treatment increased control of endometrial tumor growth in a novel HLA-A matched BLT humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4185.

Screening Peptide Drug Candidates To Neutralize Whole Viral Agents: A Case Study with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

The COVID-19 pandemic revealed the need for therapeutic and pharmaceutical molecule development in a short time with different approaches. Although boosting immunological memory by vaccination was the quickest and robust strategy, still medication is required for the immediate treatment of a patient. A popular approach is the mining of new therapeutic molecules. Peptide-based drug candidates are also becoming a popular avenue. To target whole pathogenic viral agents, peptide libraries can be employed. With this motivation, we have used the 12mer M13 phage display library for selecting SARS-CoV-2 targeting peptides as potential neutralizing molecules to prevent viral infections. Panning was applied with four iterative cycles to select SARS-CoV-2 targeting phage particles displaying 12-amino acid-long peptides. Randomly selected peptide sequences were synthesized by a solid-state peptide synthesis method. Later, selected peptides were analyzed by the quartz crystal microbalance method to characterize their molecular interaction with SARS-CoV-2's S protein. Finally, the neutralization activity of the selected peptides was probed with an in-house enzyme-linked immunosorbent assay. The results showed that scpep3, scpep8, and scpep10 peptides have both binding and neutralizing capacity for S1 protein as a candidate for therapeutic molecule. The results of this study have a translational potential with future in vivo and human studies.

Anti–Interleukin-23 Autoantibodies in Adult-Onset Immunodeficiency

BackgroundAutoantibodies against interleukin-12 (anti–interleukin-12) are often identified in patients with thymoma, but opportunistic infections develop in only some of these patients. Interleukin-12 (with subunits p40 and p35) shares a common subunit with interleukin-23 (subunits p40 and p19). In a patient with disseminated Burkholderia gladioli infection, the identification of both anti–interleukin-23 and anti–interleukin-12 prompted further investigation.MethodsAmong the patients (most of whom had thymoma) who were known to have anti–interleukin-12, we screened for autoantibodies against interleukin-23 (anti–interleukin-23). To validate the potential role of anti–interleukin-23 with respect to opportunistic infection, we tested a second cohort of patients with thymoma as well as patients without either thymoma or known anti–interleukin-12 who had unusual infections.ResultsAmong 30 patients with anti–interleukin-12 who had severe mycobacterial, bacterial, or fungal infections, 15 (50%) also had autoantibodies that neutralized interleukin-23. The potency of such neutralization was correlated with the severity of these infections. The neutralizing activity of anti–interleukin-12 alone was not associated with infection. In the validation cohort of 91 patients with thymoma, the presence of anti–interleukin-23 was associated with infection status in 74 patients (81%). Overall, neutralizing anti–interleukin-23 was detected in 30 of 116 patients (26%) with thymoma and in 30 of 36 patients (83%) with disseminated, cerebral, or pulmonary infections. Anti–interleukin-23 was present in 6 of 32 patients (19%) with severe intracellular infections and in 2 of 16 patients (12%) with unusual intracranial infections, including Cladophialophora bantiana and Mycobacterium avium complex.ConclusionsAmong patients with a variety of mycobacterial, bacterial, or fungal infections, the presence of neutralizing anti–interleukin-23 was associated with severe, persistent opportunistic infections. (Funded by the National Institute of Allergy and Infectious Diseases and others.)

Neutralizing activity of anti-respiratory syncytial virus monoclonal antibody produced in Nicotiana benthamiana

ABSTRACT Respiratory syncytial virus (RSV) is a highly contagious virus that affects the lungs and respiratory passages of many vulnerable people. It is a leading cause of lower respiratory tract infections and clinical complications, particularly among infants and elderly. It can develop into serious complications such as pneumonia and bronchiolitis. The development of RSV vaccine or immunoprophylaxis remains highly active and a global health priority. Currently, GSK’s Arexvy™ vaccine is approved for the prevention of lower respiratory tract disease in older adults (>60 years). Palivizumab and currently nirsevimab are the approved monoclonal antibodies (mAbs) for RSV prevention in high-risk patients. Many studies are ongoing to develop additional therapeutic antibodies for preventing RSV infections among newborns and other susceptible groups. Recently, additional antibodies have been discovered and shown greater potential for development as therapeutic alternatives to palivizumab and nirsevimab. Plant expression platforms have proven successful in producing recombinant proteins, including antibodies, offering a potential cost-effective alternative to mammalian expression platforms. Hence in this study, an attempt was made to use a plant expression platform to produce two anti-RSV fusion (F) mAbs 5C4 and CR9501. The heavy-chain and light-chain sequences of both these antibodies were transiently expressed in Nicotiana benthamiana plants using a geminiviral vector and then purified using single-step protein A affinity column chromatography. Both these plant-produced mAbs showed specific binding to the RSV fusion protein and demonstrate effective viral neutralization activity in vitro. These preliminary findings suggest that plant-produced anti-RSV mAbs are able to neutralize RSV in vitro.

A Potent Neutralizing Monoclonal Antibody to Human Growth Hormone Suppresses Insulin-Like Growth Factor-1 in Female Rats.

Acromegaly and gigantism are disorders caused by hypersecretion of growth hormone (GH), usually from pituitary adenomas. Although somatostatin analogues (SSA), dopamine agonists, and GH receptor antagonists are important therapeutic agents, all of these have issues with their effectiveness, safety, and/or convenience of use. To overcome these, we developed a GH-specific potent neutralizing a mouse monoclonal antibody (mAb) named 13H02. 13H02 selectively bound both to human and monkey GH with high affinity, and strongly inhibited the biological activity of GH in the Nb2 rat lymphoma cell proliferation assay. In hypophysectomized/GH-supplemented rats, a single subcutaneous administration of 13H02 significantly and dose-dependently lowered the serum insulin-like growth factor-1 levels. To pursue the therapeutic potential of this antibody for acromegaly and gigantism, we humanized 13H02 to reduce its immunogenicity and applied a single amino acid mutation in the Fc region to extend its serum half-life. The resulting antibody, Hu-13H02m, also showed GH-specific neutralizing activity, similar to the parental 13H02, and showed improved binding affinity to human FcRn.

Immune and behavioral correlates of protection against symptomatic post-vaccination SARS-CoV-2 infection.

We sought to determine pre-infection correlates of protection against SARS-CoV-2 post-vaccine inzfections (PVI) acquired during the first Omicron wave in the United States. Serum and saliva samples from 176 vaccinated adults were collected from October to December of 2021, immediately before the Omicron wave, and assessed for SARS-CoV-2 Spike-specific IgG and IgA binding antibodies (bAb). Sera were also assessed for bAb using commercial assays, and for neutralization activity against several SARS-CoV-2 variants. PVI duration and severity, as well as risk and precautionary behaviors, were assessed by questionnaires. Serum anti-Spike IgG levels assessed by research assay, neutralization titers against Omicron subvariants, and low home risk scores correlated with protection against PVIs after multivariable regression analysis. Commercial assays did not perform as well as research assay, likely due to their lower dynamic range. In the 32 participants that developed PVI, anti-Spike IgG bAbs correlated with lower disease severity and shorter duration of illness.

Comparative analysis of the efficacy of vaccines using structural protein subunits of the severe fever with thrombocytopenia syndrome virus.

The severe fever with thrombocytopenia syndrome virus (SFTSV) represents a significant emerging health threat as a tick-borne pathogen that causes SFTS, with mortality rates ranging between 10 and 30%. Despite the considerable risk presented by SFTSV, an effective vaccine has yet to be developed. Our study assessed the efficacy of recombinant protein vaccines, focusing on the purified nucleocapsid protein (NP) and surface glycoproteins (Gn and Gc), against SFTSV in both singular and combined formulations. Individual vaccinations with NP or Gn subunits yielded partial protection in type I interferon receptor-knockout (IFNAR-KO) mice, with survival rates of 66.7 and 16.7%, respectively, whereas Gc vaccination did not confer significant protection, resulting in 100% mortality similar to that of the unvaccinated control group. Notably, NP vaccination substantially enhanced antigen-specific T cell responses, and Gc vaccination exhibited strong neutralizing activity against SFTSV. Among the combined recombinant protein formulations (Gn + NP, Gc + NP, and Gn + Gc + NP) tested, the Gc + NP combination provided the highest survival rate (85.7%) following challenge with a lethal dose of SFTSV, highlighting its potential as a vaccine candidate. Longitudinal studies showed that antibody levels in both wild type C57BL/6 and IFNAR-KO mice peaked between 2 and 3 months post-vaccination and declined over time. A notable decrease in NP-specific CD8+ T cell responses was observed 6 months post-vaccination in C57BL/6 mice, while NP-specific CD4+ T cell responses persisted up to 12 months. By 12 months post-vaccination, all IFNAR-KO mice vaccinated with single subunit antigens succumbed to the virus, suggesting that effective protection against SFTS may rely on antibody responses to subunit antigens and/or CD8+ T cell activity. These findings underscore the necessity of an optimized SFTS vaccine that combines protective antigens with an adjuvant system to ensure durable humoral and cellular immunity.

Semen enhances transmitted/founder HIV-1 infection and only marginally reduces antiviral activity of broadly neutralizing antibodies.

Topically applied microbicides may play a critical role in preventing sexual transmission of human immunodeficiency virus type 1 (HIV-1); however, their efficacy can be compromised by amyloid fibrils present in semen, which significantly increase HIV-1 infectivity. This phenomenon may have contributed to the failure of most microbicide candidates in clinical settings. Understanding the impact of semen on microbicide effectiveness is thus crucial. In our study, we evaluated the influence of semen on the neutralizing activity of broadly neutralizing antibodies (bNAbs), including PG16, PGT121, 10-1074, 3BNC117, and VRC01, which are potential microbicide candidates. We found that semen enhances infection of HIV-1 transmitted/founder viruses but only marginally affects the neutralizing activity of tested antibodies, suggesting their potential for microbicide application. Our findings underscore the need to consider semen-mediated enhancement when evaluating and developing microbicides and highlight the potential of incorporating HIV-1 bNAbs in formulations to enhance efficacy and mitigate HIV-1 transmission during sexual encounters.IMPORTANCEThis study examined the impact of semen on the development of microbicides, substances used to prevent the transmission of HIV-1 during sexual activity. Semen contains certain components that can render the virus more infectious, posing a challenge to microbicide effectiveness. Researchers specifically investigated the effect of semen on a group of powerful antibodies called broadly neutralizing antibodies, which can neutralize a large spectrum of different HIV-1 variants. The results revealed that semen only had a minimal effect on the antibodies' ability to neutralize the virus. This is promising because it suggests that these antibodies could still be effective in microbicides, even in the presence of semen. Understanding this interaction is crucial for developing better strategies to prevent HIV-1 transmission. By incorporating the knowledge gained from this study, scientists can now focus on creating microbicides that consider the impact of semen, bringing us closer to more effective prevention methods.

Diminished neutralizing activity against the XBB1.5 strain in 55.9% of individuals post 6 months COVID-19 mRNA booster vaccination: insights from a pseudovirus assay on 1,353 participants in the Fukushima vaccination community survey, Japan.

This study investigates the neutralizing activity against the XBB1.5 variant and the ancestral strain in a population post-bivalent vaccination using a pseudo virus assay validated with authentic virus assay. While bivalent booster vaccination and past infections enhanced neutralization against the XBB 1.5 strain, individuals with comorbidities showed reduced responses. The study suggests the need for continuous vaccine updates to address emerging SARS-CoV-2 variants and highlights the importance of monitoring real-world immune responses.

Th1 cell immune response in Talaromyces marneffei infection with anti-interferon-γ autoantibody syndrome.

Anti-interferon-γ autoantibody (AIGA) syndrome may be the basis of disseminated Talaromyces marneffei infection in human immunodeficiency virus (HIV)-negative adults. However, the pathogenesis of Th1 cell immunity in T. marneffei infection with AIGA syndrome is unknown. A multicenter study of HIV-negative individuals with T. marneffei infection was conducted between September 2018 and September 2020 in Guangdong and Guangxi, China. Patients were divided into AIGA-positive (AP) and AIGA-negative (AN) groups according to the AIGA titer and neutralizing activity. The relationship between AIGA syndrome and Th1 immune deficiency was investigated by using AP patient serum and purification of AIGA. Fifty-five HIV-negative adults with disseminated T. marneffei infection who were otherwise healthy were included. The prevalence of AIGA positivity was 83.6%. Based on their AIGA status, 46 and 9 patients were assigned to the AP and AN groups, respectively. The levels of Th1 cells, IFN-γ, and T-bet were higher in T. marneffei-infected patients than in healthy controls. However, the levels of CD4+ T-cell STAT-1 phosphorylation (pSTAT1) and Th1 cells were lower in the AP group than in the AN group. Both the serum of patients with AIGA syndrome and the AIGA purified from the serum of patients with AIGA syndrome could reduce CD4+ T-cell pSTAT1, Th1 cell differentiation and T-bet mRNA, and protein expression. The Th1 cell immune response plays a pivotal role in defense against T. marneffei infection in HIV-negative patients. Inhibition of the Th1 cell immune response may be an important pathological effect of AIGA syndrome.IMPORTANCEThe pathogenesis of Th1 cell immunity in Talaromyces marneffei infection with anti-interferon-γ autoantibody (AIGA) syndrome is unknown. This is an interesting study addressing an important knowledge gap regarding the pathogenesis of T. marneffei in non-HIV positive patients; in particular patients with AIGA. The finding of the Th1 cell immune response plays a pivotal role in defense against T. marneffei infection in HIV-negative patients, and inhibition of the Th1 cell immune response may be an important pathological effect of AIGA syndrome, which presented in this research could help bridge the current knowledge gap.

Tethering of soluble immune effectors to mucin and chitin reflects a convergent and dynamic role in gut immunity.

The immune system employs soluble effectors to shape luminal spaces. Antibodies are soluble molecules that effect immunological responses, including neutralization, opsonization, antibody-dependent cytotoxicity and complement activation. These molecules are comprised of immunoglobulin (Ig) domains. The N-terminal Ig domains recognize antigen, and the C-terminal domains facilitate their elimination through phagocytosis (opsonization). A less-recognized function mediated by the C-terminal Ig domains of the IgG class of antibodies (Fc region) involves the formation of multiple low-affinity bonds with the mucus matrix. This association anchors the antibody molecule to the matrix to entrap potential pathogens. Even though invertebrates are not known to have antibodies, protochordates have a class of secreted molecules containing Ig domains that can bind bacteria and potentially serve a similar purpose. The VCBPs (V region-containing chitin-binding proteins) possess a C-terminal chitin-binding domain that helps tether them to chitin-rich mucus gels, mimicking the IgG-mediated Fc trapping of microbes in mucus. The broad functional similarity of these structurally divergent, Ig-containing, secreted effectors makes a case for a unique form of convergent evolution within chordates. This opinion essay highlights emerging evidence that divergent secreted immune effectors with Ig-like domains evolved to manage immune recognition at mucosal surfaces in strikingly similar ways. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

An in vivo BSL-2 model for henipavirus infection based on bioluminescence imaging of recombinant Cedar virus replication in mice.

Henipaviruses are enveloped single-stranded, negative-sense RNA viruses of the paramyxovirus family. Two henipaviruses, Nipah virus and Hendra virus, cause a systemic respiratory and/or neurological disease in humans and ten additional species of mammals, with a high fatality rate. Because of their highly pathogenic nature, Nipah virus and Hendra virus are categorized as BSL-4 pathogens, which limits the number and scope of translational research studies on these important human pathogens. To begin to address this limitation, we are developing a BSL-2 model of authentic henipavirus infection in mice, using the non-pathogenic henipavirus, Cedar virus. Notably, wild-type mice are highly resistant to Hendra virus and Nipah virus infection. However, previous work has shown that mice lacking expression of the type I interferon receptor (IFNAR-KO mice) are susceptible to both viruses. Here, we show that luciferase-expressing recombinant Cedar virus (rCedV-luc) is also able to replicate and establish a transient infection in IFNAR-KO mice, but not in wild-type mice. Using longitudinal bioluminescence imaging (BLI) of luciferase expression, we detected rCedV-luc replication as early as 10 h post-infection. Viral replication peaks between days 1 and 3 post-infection, and declines to levels undetectable by BLI by 7 days post-infection. Immunohistochemistry is consistent with viral infection and replication in endothelial cells and other non-immune cell types within tissue parenchyma. Serology analyses demonstrate significant IgG responses to the Cedar virus surface glycoprotein with potent neutralizing activity in IFNAR-KO mice, whereas antibody responses in wild-type animals were non-significant. Overall, these data suggest that rCedV-luc infection of IFNAR-KO mice represents a viable platform for the study of in vivo henipavirus replication, anti-henipavirus host responses and henipavirus-directed therapeutics.

Evaluation of Long-Term Adaptive Immune Responses Specific to SARS-CoV-2: Effect of Various Vaccination and Omicron Exposure.

The immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) becomes increasingly complex as individuals receive different combinations of vaccine doses and encounter breakthrough infections. Our study focused on the immunogenicity observed over a two-year period in healthy individuals who completed a two-dose series and then experienced booster and/or Omicron infection. In June 2023, we recruited 78 healthcare workers who had previously participated in clinical research initiated in March 2021 at a single medical center in South Korea. At 1, 5, 11, and 25 months after a second dose, we assessed SARS-CoV-2-specific humoral and cellular immune responses. Longitudinal monitoring revealed a significant decline in humoral immunity levels after the second vaccine dose, followed by a substantial increase post-third vaccination or breakthrough infection. In contrast, stable cellular immune responses were consistently observed, with peak humoral and cellular immune measures reached at 25 months after the second dose. Among infection-naïve participants, three-dose vaccinated individuals had decreased neutralizing activity against wild-type (WT) and negative activities against Omicron subvariants BA.2 and BA.4/5, whereas those who received a fourth dose of bivalent BNT had significantly increased neutralizing activity (p < 0.05). All immune metrics tended to increase as the number of vaccine doses increased. Among participants with 4-exposure, homologous vaccination (mRNA × 4) led to higher humoral immunity, whereas heterologous vaccination (ChAd × 2/mRNA × 2) induced stronger cellular responses against multiple SARS-CoV-2 variants by enzyme-linked immunospot assays (p < 0.05). Immune responses from bivalent vaccines or Omicron infection did not show statistically significant differences among exposure number-matched participants (p > 0.05). Omicron exposure significantly increased cross-neutralizing activity, but magnitude of cellular immunity was not significantly altered by Omicron exposure. Our longitudinal study highlights the evolving complexity of SARS-CoV-2 immune responses, showing enhanced immunity with multiple vaccine doses and robust cellular responses from heterologous vaccination. These findings emphasize the need for ongoing surveillance to optimize vaccination strategies against emerging variants.

Long-term analysis of humoral responses and spike-specific T cell memory to Omicron variants after different COVID-19 vaccine regimens.

The emergence of SARS-CoV-2 variants has raised concerns about the sustainability of vaccine-induced immunity. Little is known about the long-term humoral responses and spike-specific T cell memory to Omicron variants, with specific attention to BA.4/5, BQ.1.1, and XBB.1. We assessed immune responses in 50 uninfected individuals who received varying three-dose vaccination combinations (2X AstraZeneca + 1X Moderna, 1X AstraZeneca + 2X Moderna, and 3X Moderna) against wild-type (WT) and Omicron variants at eight months post-vaccination. The serum antibody titers were analyzed by enzyme-linked immunosorbent assays (ELISA), and neutralizing activities were examined by pseudovirus and infectious SARS-CoV-2 neutralization assays. T cell reactivities and their memory phenotypes were determined by flow cytometry. We found that RBD-specific antibody titers, neutralizing activities, and CD4+ T cell reactivities were reduced against Omicron variants compared to WT. In contrast, CD8+ T cell responses, central memory, effector memory, and CD45RA+ effector memory T cells remained unaffected upon stimulation with the Omicron peptide pool. Notably, CD4+ effector memory T cells even exhibited a higher proportion of reactivity against Omicron variants. Furthermore, participants who received three doses of the Moderna showed a more robust response regarding neutralization and CD8+ T cell reactions than other three-dose vaccination groups. Reduction of humoral and CD4+ T cell responses against Omicron variants in vaccinees suggested that vaccine effectiveness after eight months may not have sufficient protection against the new emerging variants, which provides valuable information for future vaccination strategies such as receiving BA.4/5 or XBB.1-based bivalent vaccines.

Efficacy of Preexposure Prophylaxis with Monoclonal Antibody Tixagevimab-Cilgavimab against Emerging SARS-CoV-2 Resistant Variants in Patients with Chronic Lymphocytic Leukemia

Introduction: Preexposure prophylaxis with monoclonal antibodies (mAbs) was developed in addition to COVID-19 vaccine for immunocompromised and those with insufficient immune response, among them patients with CLL. Omicron variant and its sublineages evolved mutations that escape mAbs neutralizing effect, yet the extent of which was not studied. Methods: We evaluated anti-spike titers and neutralization activity of COVID-19 wild-type (WT), Delta, Omicron, BA.2, BA.4, and BA.5 before and after tixagevimab-cilgavimab (TGM/CGM) dose of 150/150 mg or 300/300 mg in patients with CLL. Results: 70 patients were tested 2 weeks before and 4 weeks after receiving TGM/CGM mAbs. After TGM/CGM, anti-spike ab level increased 170-folds from 13.6 binding antibody unit (BAU)/mL (IQR, 0.4–288) to 2,328 BAU/mL (IQR, 1,681–3,500). Neutralization activity increased in all variants and was 176-folds higher in WT and 55-folds higher in Delta compared to 10-folds higher in Omicron and its sublineages (BA.2 ×11, BA.4 ×4, BA.5 ×18). Over follow-up period of 3 months, 20 patients (29%) with CLL acquired COVID-19 infection, all recovered uneventfully. In a multivariate analysis, anti-spike antibody titer was found a significant predictor for post-TGM/CGM COVID-19 infection. Conclusion: Efficacy of preexposure prophylaxis with TGM/CGM in patients with CLL is significantly reduced in era of Omicron and its sublineages BA.2, BA.4, and BA.5.

A Novel Polymer Nanoparticle Polydimethyl Diallyl Ammonium Chloride as An Adjuvant Enhances the Immune Response of SARS-CoV-2 Subunit Vaccine.

The Coronavirus Disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has a significant impact on global health and the economy. It has underscored the urgent need for a stable, easily produced and effective vaccine. This study presents a novel approach using SARS-CoV-2 spike (S) protein-conjugated nanoparticles (NPs) in combination with cyclic GMP-AMP (cGAMP) (S-NPs-cGAMP) as a subunit vaccine. When mice are immunized, the antiserum of S-NPs-cGAMP group exhibits a 16-fold increase in neutralizing activity against a pseudovirus, compared to S protein group. Additionally, S-NPs-cGAMP induces even higher levels of neutralizing antibodies. Remarkably, the vaccine also triggers a robust humoral immune response, as evidenced by a notable elevation in virus-specific IgG and IgM antibodies. Furthermore, after 42 days of immunization, there is an observed increase in specific immune cell populations in the spleen. CD3+CD4+ and CD3+CD8+T lymphocytes, as well as B220+CD19+ and CD3-CD49b+ NK lymphocytes, show an upward trend, indicating a positive cellular immune response. Moreover, the S-NPs-cGAMP demonstrates promising results against the Delta strain and exhibits good cross-neutralization potential against other variants. These findings suggest that pDMDAAC NPs is potential adjuvant and could serve as a versatile platform for future vaccine development.

Neutralizing immunity against coronaviruses in Tanzanian health care workers

The ongoing vaccination efforts and exposure to endemic and emerging coronaviruses can shape the population's immunity against this group of viruses. In this study, we investigated neutralizing immunity against endemic and emerging coronaviruses in 200 Tanzanian frontline healthcare workers (HCWs). Despite low vaccination rates (19.5%), we found a high SARS-CoV-2 seroprevalence (94.0%), indicating high exposure in these HCWs. Next, we determined the neutralization capacity of antisera against human coronavirus NL63, and 229E, SARS-CoV-1, MERS-CoV and SARS-CoV-2 (including Omicron subvariants: BA.1, BQ.1.1 and XBB.1.5) using pseudovirus neutralization assay. We observed a broad range of neutralizing activity in HCWs, but no neutralization activity detected against MERS-CoV. We also observed a strong correlation between neutralizing antibody titers for SARS-CoV-2 and SARS-CoV-1, but not between other coronaviruses. Cross-neutralization titers against the newer Omicron subvariants, BQ.1.1 and XBB.1.5, was significantly reduced compared to BA.1 and BA.2 subvariants. On the other hand, the exposed vaccinated HCWs showed relatively higher median cross-neutralization titers against both the newer Omicron subvariants and SARS-CoV-1, but did not reach statistical significance. In summary, our findings suggest a broad range of neutralizing potency against coronaviruses in Tanzanian HCWs with detectable neutralizing immunity against SARS-CoV-1 resulting from SARS-CoV-2 exposure.

Discordant Antigenic Properties of Soluble and Virion SARS-CoV-2 Spike Proteins.

Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2.

Comparison of Neutralizing Activity between Vaccinated and Unvaccinated Hospitalized COVID-19 Patients Infected with Delta, Omicron BA.1, or Omicron BA.2 Variant.

Understanding the immune response to evolving viral strains is crucial for evidence-informed public health strategies. The main objective of this study is to assess the influence of vaccination on the neutralizing activity of SARS-CoV-2 delta and omicron infection against various SARS-CoV-2 variants. A total of 97 laboratory-confirmed COVID-19 cases were included. To assess the influence of vaccination on neutralizing activity, we measured the neutralizing activity of SARS-CoV-2 delta or omicron (BA.1 or BA.2) infection against wild-type (WT), delta, BA.1, and BA.2, with the results stratified based on vaccination status. The neutralizing activity against the WT, delta, and omicron variants (BA.1 and BA.2) was significantly higher in the vaccinated patients than those in the unvaccinated patients. In the unvaccinated individuals infected with the delta variant, the decrease in binding to BA.1 and BA.2 was statistically significant (3.9- and 2.7-fold, respectively) compared to the binding to delta. In contrast, vaccination followed by delta breakthrough infection improved the cross-neutralizing activity against omicron variants, with only 1.3- and 1.2-fold decreases in BA.1 and BA.2, respectively. Vaccination followed by infection improved cross-neutralizing activity against WT, delta, and BA.2 variants in patients infected with the BA.1 variant, compared to that in unvaccinated patients. Vaccination followed by delta or BA.1 infection is associated with improved cross-neutralizing activity against different SARS-CoV-2 variants. The enhanced protection provided by breakthrough infections could have practical implications for optimizing vaccination strategies.