Structure-Activity Relationships toward the Identification of a High-Potency Selective Human Toll-like Receptor-7 Agonist.

Toll-like receptor (TLR)-7 agonists are immunostimulatory vaccine adjuvants. A systematic structure-activity relationship (SAR) study of TLR7-active 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine led to the identification of a potent hTLR7-specific p-hydroxymethyl IMDQ 23 with an EC50 value of 0.22 µM. The SAR investigation also resulted in the identification of TLR7 selective carboxamide 12 with EC50 values of 0.32 µM for hTLR7 and 18.25 µM for hTLR8. In the vaccination study, TLR7-specific compound 23 alone or combined with alum (aluminum hydroxide wet gel) showed adjuvant activity for a spike protein immunogen in mice, with enhanced anti-spike antibody production. Interestingly, the adjuvant system comprising carboxamide 12 and alum showed prominent adjuvant activity with high levels of IgG1, IgG2b, and IgG2c in immunized mice, confirming a balanced Th1/Th2 response. In the absence of any apparent toxicity, the TLR7 selective agonists in combination with alum may make a suitable vaccine adjuvant.

Developmental and reproductive safety of Advax-CpG55.2™ adjuvanted COVID-19 and influenza vaccines in mice.

SpikoGen® is a recombinant spike protein vaccine against COVID-19 that obtained marketing authorization in the Middle East on October 6th, 2021, becoming the first adjuvanted protein-based COVID-19 vaccine of its type to achieve approval. SpikoGen® vaccine utilizes a unique adjuvant Advax-CpG55.2, which comprises delta inulin and CpG55.2 oligonucleotide, a synthetic human toll-like receptor (TLR)-9 agonist. As part of a safety assessment, developmental and reproductive toxicity (DART) studies were undertaken in mice of Advax-CpG55.2 adjuvanted formulations including SpikoGen®, a H7 hemagglutinin influenza vaccine (rH7HA), the bivalent combination of SpikoGen® and rH7HA, and a next-generation quadrivalent spike protein vaccine. In the first study, vaccines were administered intramuscularly to pregnant dams on gestation days (GD) 6.5 and 12.5, and in the second two doses were given in the pre-mating period with a further two doses during gestation. The doses used in the pregnant mice were 250-1000 times the usual human doses on a weight for weight basis. Strong serum antibody responses with neutralizing activity against the relevant virus were seen in the immunized dams and also at the time of weaning in the sera of their pups, consistent with robust maternal antibody transfer. No adverse effects of any of the vaccine formulations were observed in the immunized dams or their pups. Notably, there were no adverse effects of any of the Advax-CpG55.2 adjuvanted vaccines on female mating performance, fertility, ovarian or uterine parameters, embryo-fetal or postnatal survival, fetal growth, or neurofunctional development. No evidence of antigen interference was observed when SpikoGen® vaccine was mixed and co-administered with influenza hemagglutinin vaccine to pregnant dams. Together with the strong safety profile of SpikoGen® vaccine seen in adults and children in human trials, this DART study data supports the safety of Advax-CpG55.2 adjuvanted COVID-19 and influenza vaccine in women of childbearing potential including during pregnancy.

An Advax-CpG55.2 adjuvanted recombinant hemagglutinin vaccine provides immunity against H7N9 influenza in adult and neonatal mice.

There is a major unmet need for strategies to improve the immunogenicity and effectiveness of pandemic influenza vaccines, particularly in poor responder populations such as neonates. Recombinant protein approaches to pandemic influenza offer advantages over more traditional inactivated virus approaches, as they are free of problems such as egg adaptation or need for high level biosecurity containment for manufacture. However, a weakness of recombinant proteins is their low immunogenicity. We asked whether the use of an inulin polysaccharide adjuvant (Advax) alone or combined with a TLR9 agonist (CpG55.2) would enhance the immunogenicity and protection of a recombinant hemagglutinin vaccine against H7N9 influenza (rH7HA), including in neonatal mice. Advax adjuvant induced predominantly IgG1 responses against H7HA, whereas Advax-CpG55.2 adjuvant also induced IgG2a, IgG2b and IgG3 responses, consistent with the TLR9 agonist component inducing a Th1 bias. Advax-CpG55.2 adjuvanted rH7HA induced high serum neutralizing antibody titers in adult mice. In newborns it similarly overcame immune hypo-responsiveness and enhanced serum anti-rH7HA IgG levels in 7-day-old BALB/C and C57BL/6 mice. Immunized adult mice were protected against a lethal H7N9 virus challenge. When formulated with Advax-CpG55.2 adjuvant, greater protection was seen with rH7HA than with inactivated H7 whole virus antigen. Advax-CpG55.2 adjuvanted rH7HA represents a promising influenza vaccine platform for further development.

The role of Savings and Internal Lending Communities (SILCs) in improving community-level household wealth, financial preparedness for birth, and utilization of reproductive health services in rural Zambia: a secondary analysis.

BACKGROUND: Savings and Internal Lending Communities (SILCs) are a type of informal microfinance mechanism widely adapted in Zambia. The benefits of SILCs paired with other interventions have been studied in many countries. However, limited studies have examined SILCs in the context of maternal health. This study examined the association between having access to SILCs and: 1) household wealth, 2) financial preparedness for birth, and 3) utilization of various reproductive health services (RHSs). METHODS: Secondary analysis was conducted on baseline and endline household survey data collected as part of a Maternity Waiting Home (MWH) intervention trial in 20 rural communities across seven districts of Zambia. Data from 4711 women who gave birth in the previous year (baseline: 2381 endline: 2330) were analyzed. The data were stratified into three community groups (CGs): CG1) communities with neither MWH nor SILC, CG2) communities with only MWH, and CG3) communities with both MWH and SILC. To capture the community level changes with the exposure to SILCs, different women were randomly selected from each of the communities for baseline and endline data, rather than same women being surveyed two times. Interaction effect of CG and timepoint on the outcome variables - household wealth, saving for birth, antenatal care visits, postnatal care visits, MWH utilization, health facility based delivery, and skilled provider assisted delivery - were examined. RESULTS: Interaction effect of CGs and timepoint were significantly associated only with MWH utilization, health facility delivery, and skilled provider delivery. Compared to women from CG3, women from CG1 had lower odds of utilizing MWHs and delivering at health facility at endline. Additionally, women from CG1 and women from CG2 had lower odds of delivering with a skilled provider compared to women from CG3. CONCLUSION: Access to SILCs was associated with increased MWH use and health facility delivery when MWHs were available. Furthermore, access to SILCs was associated with increased skilled provider delivery regardless of the availability of MWH. Future studies should explore the roles of SILCs in improving the continuity of reproductive health services. TRIAL REGISTRATION: NCT02620436.

Stereoisomeric Pam2CS based TLR2 agonists: synthesis, structural modelling and activity as vaccine adjuvants.

Lipopeptides including diacylated Pam2CSK4 as well as triacylated Pam3CSK4 act as ligands of toll-like receptor (TLR)-2, a promising target for the development of vaccine adjuvants. The highly investigated Pam2CSK4 and Pam3CSK4, despite their aqueous solubility have not performed well as vaccine adjuvants which may be attributable to potential denaturation of protein antigens by these cationic surfactant-like lipopeptides. In the present investigation, we synthesized (R), (S) and racemic Pam2CS(OMe) analogs and their N-acetyl derivatives without the tetralysine component to systematically investigate the effect of stereochemistry at the thio-glycerol lipopeptide core of these lipopeptide based TLR2 agonists. The resulting compounds were compared using TLR2 reporter cell-based assays and the ability of the synthesized lipopeptides to stimulate cytokine production (IL-6, IL-10 and TNF-α) by freshly collected human PBMCs and CD40 and CD86 expressions by mouse spleen cells was also investigated. Notably, few synthesized lipopeptides were found to be potent TLR2/6 agonists, inducing cytokine production and upregulating CD40 and CD86 expressions. The TLR2/6 agonistic lipopeptides were further assessed for vaccine adjuvant effects in mice. The results confirmed that the R-stereochemistry at the thio-glycerol lipopeptide core was preferred for maximal TLR2/6 activity, as reflected in Th1 immune deviation, higher antibody levels and enhanced vaccine protection against a lethal influenza challenge.

Out-of-Pocket Expenditures for Delivery for Maternity Waiting Home Users and Non-users in Rural Zambia.

BACKGROUND: Utilizing maternity waiting homes (MWHs) is a strategy to improve access to skilled obstetric care in rural Zambia. However, out-of-pocket (OOP) expenses remain a barrier for many women. We assessed delivery-related expenditure for women who used MWHs and those who did not who delivered at a rural health facility. METHODS: During the endline of an impact evaluation for an MWH intervention, household surveys (n = 826) were conducted with women who delivered a baby in the previous 13 months at a rural health facility and lived >10 km from a health facility in seven districts of rural Zambia. We captured the amount women reported spending on delivery. We compared OOP spending between women who used MWHs and those who did not. Amounts were converted from Zambian kwacha (ZMW) to US dollar (USD). RESULTS: After controlling for confounders, there was no significant difference in delivery-related expenditure between women who used MWHs (US$40.01) and those who did not (US$36.66) (P=.06). Both groups reported baby clothes as the largest expenditure. MWH users reported spending slightly more on accommodation compared to those did not use MWHs, but this difference represents only a fraction of total costs associated with delivery. CONCLUSION: Findings suggest that for women coming from far away, utilizing MWHs while awaiting delivery is not costlier overall than for women who deliver at a health facility but do not utilize a MWH.

Combined delivery of TLR2 and TLR7 agonists by Nanostructured lipid carriers induces potent vaccine adjuvant activity in mice.

Toll-like receptor (TLR) agonists are promising adjuvants and the combination of TLR agonists enhance immune responses by providing synergistic immune activity via triggering different signalling pathways. However, systematic cytotoxicity due to the immediate release of such immune potentiators from the site of injection hampers its clinical performance. Nanostructured lipid carriers (NLCs) offer a possibility to incorporate multiple TLR agonists with high encapsulation efficiency and slow drug release. Herein, we synthesized NLCs from didodecyldimethylammonium bromide (D12DAB) and oleic acid and used these to co-encapsulate a Pam2CS derivative (T-2, TLR2 agonist) with an imidazoquinoline derivative (T-7, TLR7 agonist) as a combination vaccine adjuvant. Hydrodynamic diameter and zeta potential of the prepared NLCs were found to be in the range of 200-500 nm and 23-27 mV, respectively. Spherical shape and size of prepared NLCs were also assessed through Field Emission Scanning Electron Microscopy (FE-SEM) and Transmission Electron Microscopy (TEM) analysis. In-vitro release studies of T-7 demonstrated sustained release and the addition of lipopeptide T-2 augmented encapsulation efficiency (from 84 to 92.9%) with a slight trigger in the release percentage. All NLC formulations were screened in TLR2/1, TLR2/6, TLR7 and TLR8 reporter cell lines and loaded NLC formulation showed high TLR2 and TLR7 agonistic activity. Adjuvant potency was evaluated through intramuscular immunization of female C57BL/6 mice with recombinant hepatitis B surface antigen and influenza hemagglutinin protein. T-2 and T-7 loaded NLCs induced good protective efficacy in mice challenged with a lethal dose of influenza virus.

Direct and opportunity costs related to utilizing maternity waiting homes in rural Zambia.

AIM: To assess the direct and opportunity costs involved in utilising maternity waiting homes. METHOD: A cross-sectional admission survey administered to women who used ten maternity waiting homes across two rural districts in Zambia. A total of 3,796 women participated in the survey. Descriptive analysis was conducted on three domains of the data: demographic characteristics of women, direct costs, and opportunity costs. FINDINGS: Waiting to deliver (86.3%), safe birth (70.8%), and distance (56.0%) were the most frequent reasons women reported for using a maternity waiting home. In terms of direct costs, roughly 65% of the women brought seven days or fewer days' worth of food to the maternity waiting homes, with salt, mealie meals, and vegetables being the most frequently brought items. Only 5.8% of the women spent money on transport. More than half of the women reported paying user fees that ranged from 1 to 5 or more kwacha (US$0.10- 0.52). In terms of opportunity costs, 52% of the women participated in some form of income generating activities (IGAs) when at home. Approximately 35% of the women reported they lost earned income (1 to 50 or more kwacha) by staying at a maternity waiting home. CONCLUSION: A large proportion of women paid for food and user fees to access a maternity waiting home, while a low number of women paid for transport. Even though it is difficult to assign monetary value to women's household chores, being away from these responsibilities and the potential loss of earned income appear to remain a cost to accessing maternity waiting homes. More research is needed to understand how to overcome these financial constraints and assist women in utilising a maternity waiting home.

Rapid development of analytical methods for evaluating pandemic vaccines: a COVID-19 perspective.

Vaccines are key in charting a path out of the COVID-19 pandemic. However, development of new vaccines is highly dependent on availability of analytical methods for their design and evaluation. This paper highlights the challenges presented in having to rapidly develop vaccine analytical tools during an ongoing pandemic, including the need to address progressive virus mutation and adaptation which can render initial assays unreliable or redundant. It also discusses the potential of new computational modeling techniques to model and analyze key viral proteins and their attributes to assist vaccine production and assay design. It then reviews the current range of analytical tools available for COVID-19 vaccine application, ranging from in vitro assays for immunogen characterization to assays to measure vaccine responses in vivo. Finally, it provides a future perspective for COVID-19 vaccine analytical tools and attempts to predict how the field might evolve over the next 5-10 years.

Exploring change over time in community mobilization domains: results from a maternity waiting home intervention in rural Zambia.

BACKGROUND: Community mobilization (CM) is recommended as a best practice intervention for low resource settings to reduce maternal mortality. Measurement of process outcomes are lacking and little is known about how CM impacts individuals or how community members perceive its function. Given the complex and recursive nature of CM interventions, research that describes the CM process at multiple levels is needed. This study examines change in CM domains at baseline and endline in rural Zambia. METHODS: This secondary analysis uses data from a large maternity waiting homes intervention in rural Zambia that employed CM over 3 years as part of a package of interventions. A 19-item CM survey was collected from three groups (women with babies < 1, health workers, community members; n = 1202) with focus groups (n = 76) at two timepoints from ten intervention and ten comparison sites. Factor analysis refined factors used to assess temporal change through multivariable regression. Independent covariates included time (baseline vs endline), intervention vs comparison site, group (women with babies, healthworkers, community members), and demographic variables. Interaction effects were checked for time and group for each factor. RESULTS: Final analyses included 1202 individuals from two districts in Zambia. Factor analysis maintained domains of governance, collective efficacy, self-efficacy, and power in relationships. CM domains of self-efficacy, power in relationships, and governance showed significant change over time in multivariable models. All increases in the self-efficacy factor were isolated within intervention communities (b = 0.34, p < 0.001) at endline. Between groups comparison showed the women with babies groups consistently had lower factor scores than the healthworkers or community member groups. CONCLUSIONS: Community mobilization interventions increase participation in communities to address health as a human right as called for in the 1978 Alma Ata Declaration. Grounded in empowerment, CM addresses socially prescribed power imbalances and health equity through a capacity building approach. These data reflect CM interventions function and have impact in different ways for different groups within the same community. Engaging directly with marginalized groups, using the community action cycle, and simultaneous quality improvement at the facility level may increase benefit for all groups, yet requires further testing in rural Zambia.

A M2 protein-based universal influenza vaccine containing Advax-SM adjuvant provides newborn protection via maternal or neonatal immunization.

BACKGROUND: Despite newborns being at increased risk of serious influenza infection, influenza vaccines are currently not recommended for use in infants under 6 months of age. We therefore sought to evaluate the protective efficacy in mice of an M2-based influenza vaccine (CapM2e) formulated with Advax-SM adjuvant. Vaccine protection was assessed via both passive maternal immunization and direct neonatal immunization. METHODS: For maternal transfer studies, female mice were immunized 1 week before and after mating. Blood was collected from both mother and offspring during weaning and pups were challenged when they reached 3 weeks of age with lethal doses of H1N1 and homologous reassortment influenza strain H3N2 with conserved M2. For direct immunization studies, newborns were immunized at 1 and 3 weeks of age and blood was collected prior to challenge at 4 weeks of age. RESULTS: Maternal immunization with CapM2e + Advax-SM vaccine induced high maternal M2e antibody levels that were passively transferred to their offspring and provided them with protection against both H1N1 and H3N2 influenza strains when challenged at 3 weeks of age. When used for direct immunization of neonatal mice, CapM2e + Advax-SM vaccine similarly induced high serum M2e antibody levels and protected against H1N1 and H3N2 influenza challenges with protection associated with inhibition of virus replication with a significant reduction in lung virus load in immunized pups. CONCLUSION: CapM2e + Advax-SM vaccine could be useful for protecting newborns against diverse influenza A strains, with opportunities to achieve protection by passive maternal immunization or active neonatal immunization. This data supports further development of this promising M2e-based vaccine candidate.

Role of Savings and Internal Lending Communities (SILCs) in improving household wealth and financial preparedness for birth in rural Zambia.

Savings and Internal Lending Communities (SILCs) are a type of informal microfinance mechanism adapted in many low- and middle-income countries (LMICs) to improve financial resources for poor and rural communities. Although SILCs are often paired with other health and non-health-related interventions, few studies have examined SILCs in the context of maternal health. This study examined the association between SILC participation, household wealth and financial preparedness for birth. The study also examined the association between sex and financial preparedness for birth. A secondary analysis was conducted on individual survey data collected from SILC participants in two rural districts of Zambia between October 2017 and February 2018. A convenience sample of 600 participants (Lundazi: n = 297; Mansa: n = 303) was analysed. Descriptive analyses were run to examine SILC participation and household wealth. Multiple binary logistic regression models were fit to assess the unadjusted and adjusted relationship between (1) SILC participation and household wealth, (2) SILC participation and financial preparedness for birth and (3) sex and financial preparedness for birth. The results show that SILC participation led to an average increase of 7.32 items of the 13 household wealth items. SILC participants who had their most recent childbirth after joining SILCs were more likely to be financially prepared for birth [adjusted odds ratio (AOR): 2.99; 95% confidence interval (95% CI): 1.70-5.26; P < 0.001] than participants who had their most recent childbirth before joining SILCs. Females were more likely to be financially prepared for birth than males if they had their most recent birth before joining an SILC (AOR: 1.79; 95% CI: 1.16-2.66; P < 0.01). SILC participation is shown to increase household wealth and financial preparedness for birth for both men and women. SILCs are a promising intervention that can help poor and rural populations by increasing financial resources and financially preparing parents for birth.

BBIQ, a pure TLR7 agonist, is an effective influenza vaccine adjuvant.

Better adjuvants are needed for vaccines against seasonal influenza. TLR7 agonists are potent activators of innate immune responses and thereby may be promising adjuvants. Among the imidazoquinoline compounds, 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (BBIQ) was reported to be a highly active TLR7 agonist but has remained relatively unexplored because of its commercial unavailability. Indeed, in silico molecular modeling studies predicted that BBIQ had a higher TLR7 docking score and binding free energy than imiquimod, the gold standard TLR7 agonist. To circumvent the availability issue, we developed an improved and higher yield method to synthesize BBIQ. Testing BBIQ on human and mouse TLR7 reporter cell lines confirmed it to be TLR7 specific with significantly higher potency than imiquimod. To test its adjuvant potential, BBIQ or imiquimod were admixed with recombinant influenza hemagglutinin protein and administered to mice as two intramuscular immunizations 2 weeks apart. Serum anti-influenza IgG responses assessed by ELISA 2 weeks after the second immunization confirmed that the mice that received vaccine admixed with BBIQ had significantly higher anti-influenza IgG1 and IgG2c responses than mice immunized with antigen alone or admixed with imiquimod. This confirmed BBIQ to be a TLR7-specific adjuvant able to enhance humoral immune responses.

Neonatal vaccine effectiveness and the role of adjuvants.

Introduction: Neonates are less responsive to vaccines than adults, making it harder to protect newborns against infection. Neonatal differences in antigen-presenting cell, B and T cell function, all likely contribute. A key question is whether novel adjuvants might be able to make neonatal vaccines more effective. Areas covered: This review addresses the issues of how to improve neonatal vaccines, which we have defined as vaccines given in the first 4 weeks of life in a human infant or the first week of life in a mouse. A search was performed using keywords including 'neonatal immunity', 'neonatal immunisation', 'vaccine' and 'adjuvant' of PubMed articles published between 1960 and 2018. Expert opinion: Sugar-like structures have recently been shown to prime the infant adaptive immune system to respond to vaccines, being potentially more effective than traditional adjuvants. Sugar-based compounds with beneficial adjuvant effects in neonatal vaccine models include delta inulin (Advax), curdlan, and trehalose 6,6'-dibehenate. Such compounds make interesting neonatal adjuvant candidates, either used alone or in combination with traditional innate immune adjuvants.

Highly conserved influenza T cell epitopes induce broadly protective immunity.

Influenza world-wide causes significant morbidity and mortality annually, and more severe pandemics when novel strains evolve to which humans are immunologically naïve. Because of the high viral mutation rate, new vaccines must be generated based on the prevalence of circulating strains every year. New approaches to induce more broadly protective immunity are urgently needed. Previous research has demonstrated that influenza-specific T cells can provide broadly heterotypic protective immunity in both mice and humans, supporting the rationale for developing a T cell-targeted universal influenza vaccine. We used state-of-the art immunoinformatic tools to identify putative pan-HLA-DR and HLA-A2 supertype-restricted T cell epitopes highly conserved among > 50 widely diverse influenza A strains (representing hemagglutinin types 1, 2, 3, 5, 7 and 9). We found influenza peptides that are highly conserved across influenza subtypes that were also predicted to be class I epitopes restricted by HLA-A2. These peptides were found to be immunoreactive in HLA-A2 positive but not HLA-A2 negative individuals. Class II-restricted T cell epitopes that were highly conserved across influenza subtypes were identified. Human CD4+ T cells were reactive with these conserved CD4 epitopes, and epitope expanded T cells were responsive to both H1N1 and H3N2 viruses. Dendritic cell vaccines pulsed with conserved epitopes and DNA vaccines encoding these epitopes were developed and tested in HLA transgenic mice. These vaccines were highly immunogenic, and more importantly, vaccine-induced immunity was protective against both H1N1 and H3N2 influenza challenges. These results demonstrate proof-of-principle that conserved T cell epitopes expressed by widely diverse influenza strains can induce broadly protective, heterotypic influenza immunity, providing strong support for further development of universally relevant multi-epitope T cell-targeting influenza vaccines.

Investigation of the biodistribution, breakdown and excretion of delta inulin adjuvant.

Insoluble, nanostructured delta inulin particles enhance the immunogenicity of co-administered protein antigens and consequently are used as a vaccine adjuvant (Advax™). To better understand their immunomodulatory properties, the in vitro hydrolysis and in vivo distribution of delta inulin particles were investigated. Delta inulin particle hydrolysis under bio-relevant acidic conditions resulted in no observable change to the bulk morphology using SEM, and HPLC results showed that only 6.1% of the inulin was hydrolysed over 21days. However, 65% of the terminal glucose groups were released, showing that acid hydrolysis relatively rapidly releases surface bound chemistries. This was used to explain in vivo biodistribution results in which delta inulin particles surface-labelled with fluorescein-5-thiosemicabizide were administered to mice using intramuscular (I.M.) or subcutaneous (S.C.) routes. Comparison analysis of the fluorescence of soluble inulin in the supernatants of homogenised tissues maintained at room temperature or heated to 100°C to solubilise particulate inulin was used to distinguish between fluorescent probe on soluble inulin and probe bound to inulin within particles. Following both I.M. and S.C. injection delta inulin exhibited a depot behaviour with local injection site residence for several weeks. Over this time, as injection site inulin reduced, there was measurable transport of intact delta inulin particles by macrophages to secondary lymphoid organs and the liver. Ultimately, the injected delta inulin became solubilised resulting in its detection in the plasma and in the urine. Thus injected delta inulin particles are initially taken up by macrophages at the site of injection, trafficked to secondary lymphoid tissue and the liver, and hydrolysed resulting in their becoming soluble and diffusing into the blood stream, from whence they are glomerularly filtered and excreted into the urine. These results provide important insights into the biodistribution of I.M. or S.C. injected delta inulin particles when used as vaccine adjuvants and their method of excretion.

Influenza immunization during pregnancy: Benefits for mother and infant.

The serious consequences of influenza infection during pregnancy have been recognized for almost a century. In this article, we reviewed the evidence on the immunogenicity, safety and impact of maternal influenza immunization for both mother and child. After vaccination, pregnant women have similar protective titers of anti-influenza antibodies as non-pregnant women, demonstrating that pregnancy does not alter the trivalent inactivated influenza vaccine immune response. Studies from the United States, Europe and resource-constrained regions demonstrate that maternal vaccination is associated with increased anti-influenza antibody concentrations and protection in the newborn child as well as the immunized mother. Given the acceptable safety profile of influenza vaccines and the World Health Organization's recommendation for its use in pregnant women, maternal vaccination with inactivated influenza vaccine is a cost-effective approach to decrease influenza disease in newborns. However, as seen for influenza immunization in the elderly, the protective efficacy of current inactivated vaccines in protection of newborns is 50% at best, indicating significant room for vaccine improvement, which could potentially be achieved by addition of a safe and effective adjuvant. Thus, global deployment of inactivated influenza immunization during pregnancy would have substantial and measurable health benefits for mothers and their newborns.

A Subset of Protective γ9δ2 T Cells Is Activated by Novel Mycobacterial Glycolipid Components.

γ9δ2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded γ9δ2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection. Based on this premise, we have been searching for M. tuberculosis antigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2 T cells. Our screening strategy includes the identification of M. tuberculosis fractions that expand γ9δ2 T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosis whole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosis antigens inducing protective γ9δ2 T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosis whole-cell lysates, suggesting that a polysaccharide was required for biological activity. Extraction of MtbWL with chloroform-methanol-water (10:10:3) resulted in a polar lipid fraction with highly enriched specific activity; this activity was further enriched by silica gel chromatography. A combination of mass spectrometry and nuclear magnetic resonance analysis of bioactive fractions indicated that 6-O-methylglucose-containing lipopolysaccharides (mGLP) are predominant components present in this active fraction. These results have important implications for the development of new immunotherapeutic approaches for prevention and treatment of TB.

Evidence for Persistence of Ectromelia Virus in Inbred Mice, Recrudescence Following Immunosuppression and Transmission to Naïve Mice.

Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status.

Functional Heterogeneity and Antimycobacterial Effects of Mouse Mucosal-Associated Invariant T Cells Specific for Riboflavin Metabolites.

Mucosal-associated invariant T (MAIT) cells have a semi-invariant TCR Vα-chain, and their optimal development is dependent upon commensal flora and expression of the nonpolymorphic MHC class I-like molecule MR1. MAIT cells are activated in an MR1-restricted manner by diverse strains of bacteria and yeast, suggesting a widely shared Ag. Recently, human and mouse MR1 were found to bind bacterial riboflavin metabolites (ribityllumazine [RL] Ags) capable of activating MAIT cells. In this study, we used MR1/RL tetramers to study MR1 dependency, subset heterogeneity, and protective effector functions important for tuberculosis immunity. Although tetramer(+) cells were detected in both MR1(+/+) and MR1(-/-) TCR Vα19i-transgenic (Tg) mice, MR1 expression resulted in significantly increased tetramer(+) cells coexpressing TCR Vß6/8, NK1.1, CD44, and CD69 that displayed more robust in vitro responses to IL-12 plus IL-18 and RL Ag, indicating that MR1 is necessary for the optimal development of the classic murine MAIT cell memory/effector subset. In addition, tetramer(+) MAIT cells expressing CD4, CD8, or neither developing in MR1(+/+) Vα19i-Tg mice had disparate cytokine profiles in response to RL Ag. Therefore, murine MAIT cells are considerably more heterogeneous than previously thought. Most notably, after mycobacterial pulmonary infection, heterogeneous subsets of tetramer(+) Vα19i-Tg MAIT cells expressing CXCR3 and α4ß1 were recruited into the lungs and afforded early protection. In addition, Vα19iCα(-/-)MR(+/+) mice were significantly better protected than were Vα19iCα(-/-)MR1(-/-), wild-type, and MR1(-/-) non-Tg mice. Overall, we demonstrate considerable functional diversity of MAIT cell responses, as well as that MR1-restricted MAIT cells are important for tuberculosis protective immunity.