Wilfried A. A. Saron

NKT cells are innate-like T cells, recruited to the skin during viral infection, yet their contributions to long-term immune memory to viruses are unclear. We identified granzyme K, a product made by cytotoxic cells including NKT cells, as linked to induction of Th1-associated antibodies during primary dengue virus (DENV) infection in humans. We examined the role of NKT cells in vivo using DENV-infected mice lacking CD1d-dependent (CD1d ^dep ) NKT cells. In CD1d-KO mice, Th1-polarized immunity and infection resolution were impaired, which was dependent on intrinsic NKT cell production of IFN-γ, since it was restored by adoptive transfer of WT but not IFN-γ–KO NKT cells. Furthermore, NKT cell deficiency triggered immune bias, resulting in higher levels of Th2-associated IgG1 than Th1-associated IgG2a, which failed to protect against a homologous DENV rechallenge and promoted antibody-dependent enhanced disease during secondary heterologous infections. Similarly, Th2 immunity, typified by a higher IgG4/IgG3 ratio, was associated with worsened human disease severity during secondary infections. Thus, CD1d ^dep NKT cells establish Th1 polarity during the early innate response to DENV, which promotes infection resolution, memory formation, and long-term protection from secondary homologous and heterologous infections in mice, with consistent associations observed in humans. These observations illustrate how early innate immune responses during primary infections can influence secondary infection outcomes.

Zika virus (ZIKV) is a mosquito-borne flavivirus that can vertically transmit from mother to fetus, potentially causing congenital defects, including microcephaly. It is not fully understood why some fetuses experience severe complications after in utero exposure to ZIKV, whereas others do not. Given the antigenic similarity between ZIKV and the closely related virus dengue (DENV) and the potential of DENV-specific antibodies to enhance ZIKV disease severity in mice, we questioned whether maternal DENV immunity could influence fetal outcomes in a nonhuman primate model of ZIKV vertical transmission. We found significantly increased severity of congenital Zika syndrome (CZS) in fetuses of DENV-immune cynomolgus macaques infected with ZIKV in early pregnancy compared with naïve controls, which occurred despite no effect on maternal ZIKV infection or antibody responses. Ultrasound measurements of head circumference and biparietal diameter measurements taken sequentially throughout pregnancy demonstrated CZS in fetuses of DENV-immune pregnant macaques. Furthermore, severe CZS enhanced by DENV immunity was typified by reduced cortical thickness and increased frequency of neuronal death, hemorrhaging, cellular infiltrations, calcifications, and lissencephaly in fetal brains. This study shows that maternal immunity to DENV can worsen ZIKV neurological outcomes in fetal primates, and it provides an animal model of vertical transmission closely approximating human developmental timelines that could be used to investigate severe ZIKV disease outcomes and interventions in fetuses.

Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in severely ill patients and the pathophysiology of disease is thought to be immune-mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens, often promoting inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and non-human primates. Using a mouse model of MC-deficiency, MC-dependent interstitial pneumonitis, hemorrhaging, and edema in the lung were observed during SARS-CoV-2 infection. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype in severe disease. MC activation in humans was confirmed, through detection of MC-specific proteases, including chymase, levels of which were significantly correlated with disease severity and with biomarkers of vascular dysregulation. These results support the involvement of MCs in lung tissue damage during SARS-CoV-2 infection in animal models and the association of MC activation with severe COVID-19 in humans, suggesting potential strategies for intervention.

COVID-19 vaccines used in humans are highly effective in limiting disease and death caused by the SARS-CoV-2 virus, yet improved vaccines that provide greater protection at mucosal surfaces, which could reduce break-through infections and subsequent transmission, are still needed. Here we show that intranasal (I.N.) vaccination with the receptor binding domain of Spike antigen of SARS-CoV-2 (S-RBD) in combination with the mucosal adjuvant mastoparan-7 improved systemic T cell responses compared to an equivalent dose of antigen delivered by the sub-cutaneous (S.C.) route, adjuvanted by either M7 or the gold-standard adjuvant, alum. T cell phenotypes induced by I.N. vaccine administration included enhanced polyfunctionality (combined IFN-γ and TNF expression) and greater numbers of T central memory (T _CM ) cells. These phenotypes were T cell-intrinsic and could be recalled in the lungs and/or brachial LNs upon antigen challenge after adoptive T cell transfer to naïve recipients. Furthermore, mucosal vaccination induced antibody responses that were similarly effective in neutralizing the binding of the parental strain of S-RBD to its ACE2 receptor, but showed greater cross-neutralizing capacity against multiple variants of concern (VOC), compared to S.C. vaccination. These results highlight the role of nasal vaccine administration in imprinting an immune profile associated with long-term T cell retention and diversified neutralizing antibody responses, which could be applied to improve vaccines for COVID-19 and other infectious diseases.

Mosquito blood-feeding behavior is a key determinant of the epidemiology of dengue viruses (DENV), the most-prevalent mosquito-borne viruses. However, despite its importance, how DENV infection influences mosquito blood-feeding and, consequently, transmission remains unclear. Here, we developed a high-resolution, video-based assay to observe the blood-feeding behavior of Aedes aegypti mosquitoes on mice. We then applied multivariate analysis on the high-throughput, unbiased data generated from the assay to ordinate behavioral parameters into complex behaviors. We showed that DENV infection increases mosquito attraction to the host and hinders its biting efficiency, the latter resulting in the infected mosquitoes biting more to reach similar blood repletion as uninfected mosquitoes. To examine how increased biting influences DENV transmission to the host, we established an in vivo transmission model with immuno-competent mice and demonstrated that successive short probes result in multiple transmissions. Finally, to determine how DENV-induced alterations of host-seeking and biting behaviors influence dengue epidemiology, we integrated the behavioral data within a mathematical model. We calculated that the number of infected hosts per infected mosquito, as determined by the reproduction rate, tripled when mosquito behavior was influenced by DENV infection. Taken together, this multidisciplinary study details how DENV infection modulates mosquito blood-feeding behavior to increase vector capacity, proportionally aggravating DENV epidemiology. By elucidating the contribution of mosquito behavioral alterations on DENV transmission to the host, these results will inform epidemiological modeling to tailor improved interventions against dengue. Significance Because dengue viruses are spread by mosquitoes during biting, transmission capacity depends on mosquito-biting behavior. For this reason, it is critical to understand how infection in mosquitoes influences biting. To answer this question, we deployed a multidisciplinary approach including high-resolution, multivariate biting behavior monitoring on mice, in vivo transmission assay, and mathematical modeling. We demonstrated that infected mosquitoes are more attracted to mice and bite more often to get the same amount of blood as uninfected mosquitoes. While the effect of increased attraction to host on transmission capacity is trivial, we showed that increased number of bites results in successive transmission. Eventually, we calculated that the infection-induced behavior changes tripled transmission capacity of mosquitoes.

Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in severely ill patients and the pathophysiology of disease is thought to be immune-mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens, often promoting inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and non-human primates. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype. MC activation in humans was confirmed, through detection of the MC-specific protease, chymase, levels of which were significantly correlated with disease severity. These results support the association of MC activation with severe COVID-19, suggesting potential strategies for intervention.

Sub-neutralizing concentrations of antibodies in dengue infected patients is a major risk factor for the development of dengue hemorrhagic fever and dengue shock syndrome. Here, we describe a mouse model with a deficiency in mast cells (MCs) in addition to a deficiency in Type-I and II IFN receptors for studying dengue virus (DENV) infection. We used this model to understand the influence of MCs in a maternal antibody-dependent model of severe dengue, where offspring born to DENV-immune mothers are challenged with a heterologous DENV serotype. Mice lacking both MCs and IFN receptors were found susceptible to primary DENV infection and showed morbidity and mortality. When these mice were immunized, pups born to DENV-immune mothers were found to be protected for a longer duration from a heterologous DENV challenge. In the absence of MCs and type-I interferon signaling, IFN-γ was found to protect pups born to naïve mothers but had the opposite effect on pups born to DENV-immune mothers. Our results highlight the complex interactions between MCs and IFN-signaling in influencing the role of maternal antibodies in DENV-induced disease severity.

Dengue virus (DENV), an arbovirus, strongly activates mast cells (MCs), which are key immune cells for pathogen immune surveillance. In animal models, MCs promote clearance of local peripheral DENV infections but, conversely, also promote pathological vascular leakage when widely activated during systemic DENV infection. Since DENV is a human pathogen, we sought to ascertain whether a similar phenomenon could occur in humans by characterizing the products released by human MCs (huMCs) upon direct (antibody-independent) DENV exposure, using the phenotypically mature huMC line, ROSA. DENV did not productively infect huMCs but prompted huMC release of proteases and eicosanoids and induced a Th1-polarized transcriptional profile. In co-culture and trans-well systems, huMC products activated human microvascular endothelial cells, involving transcription of vasoactive mediators and increased monolayer permeability. This permeability was blocked by MC-stabilizing drugs, or limited by drugs targeting certain MC products. Thus, MC stabilizers are a viable strategy to limit MC-promoted vascular leakage during DENV infection in humans.

Maternal IgE activates fetal mast cells: Mast cells (MCs) are immune cells that participate in allergic reactions through their activation by immunoglobulin E (IgE) antibodies. MCs arise early during mammalian development, but it is unclear whether IgE-mediated activation occurs in fetal tissues and what the source of IgE stimulation is. Msallam et al. show that human and mouse fetal MCs can be sensitized by IgE of maternal origin, which crosses the placental barrier through the fetal neonatal Fc receptor (see the Perspective by Rothenberg). Prenatal maternal sensitization conferred transient allergen sensitivity after birth and resulted in the development of postnatal skin and airway inflammation in the offspring after their first exposure to allergen. Thus, both maternal IgE and fetal MCs may influence mother-to-child transmission of allergic disease during gestation. Science , this issue p. 941 ; see also p. 907

Infection of fetuses with Zika virus during pregnancy and congenital defects are enhanced by maternal immunity to dengue virus.