Comparison of percutaneous vs oral infection of hamsters with the hookworm Ancylostoma ceylanicum: Parasite development, pathology and primary immune response

Background Hundreds of millions of people in poor countries continue to suffer from disease caused by bloodfeeding hookworms. While mice and rats are not reliably permissive hosts for any human hookworm species, adult Golden Syrian hamsters are fully permissive for the human and animal pathogen Ancylostoma ceylanicum. Similar to humans, hamsters may be infected with A. ceylanicum third-stage larvae orally or percutaneously. Oral infection typically leads to consistent worm yields in hamsters but may not accurately reflect the clinical and immunological manifestations of human infection resulting from skin penetration. Methodology/Principal findings In this study we compared host responses following percutaneous infection to those utilizing an established oral infection protocol. Infected hamsters exhibited a dose-dependent pathology, with 1000 percutaneous larvae (L3) causing anemia and adult worm recovery comparable to that of 50 orally administered L3. A delayed arrival and maturity of worms in the intestine was observed, as was variation in measured cellular immune responses. A long-term study found that the decline in blood hemoglobin was more gradual and did not reach levels as low, with the nadir of disease coming later in percutaneously infected hamsters. Both groups exhibited moderate growth delay, an effect that was more persistent in the percutaneously infected group. Fecal egg output also peaked later and at lower levels in the percutaneously infected animals. In contrast to orally infected hamsters, antibody titers to larval antigens continued to increase throughout the course of the experiment in the percutaneous group. Conclusions/Significance These results demonstrate that the route of infection with A. ceylanicum impacts disease pathogenesis, as well as humoral and cellular immune responses in an experimental setting. These data further validate the utility of the Golden Syrian hamster as a model of both oral and percutaneous infection with human hookworms.

Adjuvant Activity of CpG-Oligonucleotide Administered Transcutaneously in Combination with Vaccination Using a Self-Dissolving Microneedle Patch in Mice

In this study, we investigated the mechanism of transcutaneous adjuvant activity of the CpG-oligonucleotide (K3) in mice. Transcutaneous immunization (TCI) with an ovalbumin-loaded self-dissolving microneedle patch (OVA-sdMN) and K3-loaded hydrophilic gel patch (HG) increased OVA-specific Th2- and Th1-type IgG subclass antibody titers more rapidly and strongly than those after only OVA-sdMN administration. However, the antigen-specific proliferation of OVA-specific CD4+ T cells was similar between the OVA-only and the OVA+K3 groups. Population analysis of various immune cells in draining lymph nodes (dLNs) in the primary immune response revealed that the OVA+K3 combination doubled the number of dLN cells, with the most significant increase in B cells. Phenotypic analysis by flow cytometry revealed that B-cell activation and maturation were promoted in the OVA+K3 group, suggesting that direct B-cell activation by K3 largely contributed to the rapid increase in antigen-specific antibody titer in TCI. In the secondary immune response, a significant increase in effector T cells and effector memory T cells, and an increase in memory B cells were observed in the OVA+K3 group compared with that in the OVA-only group. Thus, K3, as a transcutaneous adjuvant, can promote the memory differentiation of T and B cells.

Activation of mucosal immunity and novel prophylactic and therapeutic strategy in combating COVID-19

Coronavirus disease 2019 (COVID-19) emerges as an expeditiously growing pandemic, in the human population caused by the highly transmissible RNA virus severe acute respiratory syndrome of coronavirus 2 (SARS-CoV-2). Prognosis of SARS-CoV-2 infection predominantly occurs at the angiotensin-converting enzyme 2 receptor and transmembrane protease serine type 2 positive (ACE2 + TMPRSS2)+ epithelial cells of the mucosal surface like nasal, oral mucosae, and/or the conjunctival surface of the eye where it has interacted along with the immune system. The primary host response towards the pathogen starts from an immune microenvironment of nasopharynx-associated lymphoid tissue (NALT) and mucosa-associated lymphoid tissue (MALT). The presence of exhausted lymphocytes, lymphopenia, pneumonia and cytokine storm is the hallmark of COVID-19. The multifaceted nature of co-morbidity factors like obesity and type 2 diabetes and its effects on immunity can alter the pathogenesis of SARS-CoV-2 infection. Adipose tissue is a crucial endocrine organ that secretes a plethora of factors like adipokines, cytokines, and chemokines that have a profound impact on metabolism and augments the expression of mucosal pro-inflammatory cytokines, like tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and the interleukin-12 (IL-12)/IL-23. Mucosal immunization could be a superior approach to activate mucosal and systemic immune responses against pathogenic invasion at mucosal surface entry ports. Mucosal vaccines are also able to generate strong systemic humoral immunity—required to neutralize any virus particle that dodges the primary immune response. To develop an efficient vaccine against mucosal pathogens, considering the designing of the delivery route, immunomodulatory features, and adjuvants are very important. In this article, we further provide evidence to understand the significant role of mucosal immunity, along with secretory and circulating immunoglobulin A (IgA) antibodies in generating a novel mucosal vaccine against COVID-19. Moreover, along with mucosal vaccines, we should look for combination treatment strategies with plant bioactive molecules. Glycan-binding lectins against viral proteins for targeted activation of mucosal immune response are one of such examples. This might play a promising role to halt this emerging virus.

Transplantation and Re-transplantation of Mouse Kidney Grafts to Study Local Immune Responses

Mouse kidney transplantation is widely used to study the immune response to allogeneic grafts. This response includes a circulating systemic compartment and a resident non circulating one. A distinction between these compartments remains an important caveat to the interpretation of the resident or local immune response’s importance and function. Here, we describe re-transplantation as a method to functionally test the resident component of the primary immune response while also studying the secondary recipient’s response. Our detailed, stepwise protocol can be reliably replicated for both the primary and secondary, donor and recipient operations. The techniques in this protocol can be efficiently implemented by an individual proficient in mouse kidney transplantation surgical procedures.

Transplantation and Re-transplantation of Mouse Kidney Grafts to Study Local Immune Responses

Mouse kidney transplantation is widely used to study the immune response to allogeneic grafts. This response includes a circulating systemic compartment and a resident non circulating one. A distinction between these compartments remains an important caveat to the interpretation of the resident or local immune response’s importance and function. Here, we describe re-transplantation as a method to functionally test the resident component of the primary immune response while also studying the secondary recipient’s response. Our detailed, stepwise protocol can be reliably replicated for both the primary and secondary, donor and recipient operations. The techniques in this protocol can be efficiently implemented by an individual proficient in mouse kidney transplantation surgical procedures.

Varicella-Zoster Virus (Varicella and Shingles)

A live attenuated vaccine against varicella (later also used to prevent zoster) was developed in 1974 by Takahashi and colleagues. Varicella vaccine was licensed for universal immunization of healthy children in the United States in 1995. It is also now used for this purpose in at least 15 additional countries all over the world. Varicella is disappearing in the US. Varicella vaccine has proven extremely safe and side effects are unusual, mild, and less serious than varicella or its complications. 85% of children are protected completely after 1 dose; the 15% who develop varicella despite immunization usually (but not always) have mild infections. These 15%, however, can transmit the wild type virus to others. Therefore, for optimal effect, 2 doses are required, mostly to address children who did not have an optimal primary immune response after the first dose. Waning immunity does not seem to pose a serious problem, but surveillance of vaccinees is continuing. It was demonstrated in 2005 that at a high dose of vaccine – 15 times higher than that used for prevention of varicella in children - zoster in adults can also be safely prevented. The live attenuated zoster vaccine is effective in approximately 50% of healthy individuals over age 60 who have had varicella in the past, and therefore have latent infection with varicella-zoster virus. It is given as one dose, but its effect runs out about 8 years after vaccination. In 2017, a new vaccine against zoster was also introduced. This is a subunit vaccine which does not contain contagious virus. It is even more effective than the older zoster vaccine and is over 95% effective in adults 50–≥70 years of age in preventing zoster and post herpetic neuralgia.

269 Utilizing serum proteome to understand response and resistance to immune checkpoint inhibitors in advanced non-small cell lung cancer

BackgroundPredictive biomarkers are more in need considering that responses and resistance to immune checkpoint inhibition (ICI) are poorly understood. We used a validated serum-based proteomic test, Primary Immune Response (PIR), to predict response and Protein Set Enrichment Analysis (PSEA) scores to elucidate mechanisms of early resistance to ICI in patients with advanced non-small cell lung cancer (NSCLC). (Muller et al., 2020)MethodsSerum of 43 consented NSCLC patients was collected prospectively at 2 timepoints: baseline (prior to ICI initiation) and 3 weeks after ICI initiation (median 22 days). Blinded samples were classified by the PIR test. Clinical response was evaluated using RECIST. Outcomes, including progression-free survival (PFS) and overall survival (OS), were analyzed by PIR classifications as intermediate/sensitive (not resistant) vs. resistant at baseline and 3 weeks. Multivariable regression was performed. PSEA scores indicating activity of 10 processes of interest (e.g., Type 1 immunity (Th1), complement, interferon-gamma (IFNγ)) were compared between PIR groups.ResultsOf the 43 patients, 28 received chemotherapy with ICI (chemo+ICI) and 15 received ICI alone. 31 of 43 patients (72%) were treatment naïve at baseline blood draw. PIR-resistant patients had worse survival compared to patients classified as not resistant (HR, 10.4; 95%CI, 1.3–81 ; P = 0.025). OS was also significantly lower for patients with PIR resistant result at 3 weeks (HR, 9.1; 95%CI, 1.2–72 ; P = 0.036). The difference in survival between PIR classification groups was consistently observed in the treatment naïve patients treated with chemo+ICI (log rank P = 0.02). No significant differences were observed in PFS. Clinical and pathologic characteristics, including PD-L1 expression, were not significantly associated with PIR result. In multivariable analysis including performance status, line of therapy, and PD-L1 status, PIR resistant remained a significant negative prognostic factor (HR, 8.2; 95%CI, 1.01–67; P = 0.049). PSEA scores at baseline and 3 weeks after ICI initiation showed significantly higher levels of complement, IFNγ, Th1, immune tolerance, and a lower level of wound healing (all P<0.0001) in PIR-resistant vs. PIR-intermediate/sensitive.ConclusionsThese data further validate the utility of the PIR test in predicting patient survival on ICI. Processes associated with PIR resistant result included activation of complement, IFNγ, Th1, and immune tolerance, elucidating early mechanisms of resistance to ICI in a clinical cohort.

25 Potential role of serum proteome in predicting immune-related adverse events from immunotherapy in non-small cell lung cancer

BackgroundPredicting immune-related adverse events (irAEs) in early stage is being emphasized even more. Host response to disease is reflected in serum proteome level and that allows serum proteome level as a new marker to explore response to immunotherapy. With the help of a serum-based proteomics test, Primary Immune Response (PIR), we are accessing the correlations between developing irAEs and immunotherapy in non-small cell lung cancer (NSCLC) patients.MethodsData of 48 consented NSCLC patients with baseline PIR test done within one week prior to the start of immunotherapy were collected. Samples were grouped into either sensitive or intermediate/resistant (not sensitive) by PIR classification. We analyzed the durations from the immunotherapy initiation to the first episode of irAE. IrAEs were graded according to Common Terminology Criteria for Adverse Events (CTCAE) v5.0.ResultsAmong the 48 NSCLC patients, 19 patients (39%) experienced one or more irAEs with the majority classified as either grade 1 (n=7, 36%) or grade 2 (n=10, 52%). PIR-sensitive group showed no difference in irAE free period compared to PIR-not sensitive (p=0.92, HR=0.95, 95% CI=00.3212 to 2.834). The median ‘Time to first irAE’ were undefined and 24 in PIR-sensitive and PIR-not sensitive, respectively.ConclusionsOur results demonstrated PIR-sensitive patients are not likely to tolerate immunotherapy longer without developing irAEs.

Inhibition of Anthranilate Synthase Component II, a Novel Protein of S. pneumoniae as a Potential Target for Therapy

Streptococcus pneumoniae infects the human body primarily through the respiratory tract; however, no evident inflammatory responses are observed upon infection. Even though the inflammatory response is the body's primary immune response, the latency of the inflammatory responses may be attributable to the presence of an anthranilate derivative, quinolone, an isostere of salicylic acid, which acts to suppress inflammation. The reduced immune response promotes the formation of the S. pneumoniae biofilm and increases virulence via quinolone and the derivative, fenamic acid, to elicit different responses. It was found in this study that coumarin binds with good affinity to the binding site of anthranilate synthase component II and also confers a good heme-protectant property. The enzyme anthranilate synthase is a virulent factor of S. pneumoniae and influences the inflammatory response signaling pathways. Inhibition of the anthranilate synthase pathway terminates the virulence of S. pneumoniae and helps prevent the impending severe pathogenesis of infection.

Virtual memory T cells orchestrate extralymphoid responses conducive to resident memory

A primary immune response is initiated in secondary lymphoid organs. Virtual memory CD8+ T (TVM) cells are antigen-inexperienced T cells of a central memory phenotype, acquired through self-antigen–driven homeostatic proliferation. Unexpectedly, we find that TVM cells are composed of CCR2+ and CCR2− subsets that differentially elaborate a spectrum of effector- and memory-poised functions directly in the tissue. During a primary influenza infection, TVM cells rapidly infiltrate the lungs in the first day after infection and promote early viral control. TVM cells that recognize viral antigen are retained in the tissue, clonally expand independent of secondary lymphoid organs, and give rise to tissue-resident memory cells. By orchestrating an extralymphoid primary response, heterogenous TVM cells bridge innate reaction and adaptive memory directly in the infected tissue.