Enhanced Antibody Responses Research Articles

Melatonin enhances responsiveness to Dichelobacter nodosus vaccine in sheep and increases peripheral blood CD4 T lymphocytes and IgG-expressing B lymphocytes

The immunomodulatory functions mediated by melatonin support its use as vaccine adjuvant. Previously, we have demonstrated that melatonin enhances antibody responses in sheep vaccinated against Dichelobacter nodosus. Here, we analyze the effect of melatonin on T and B lymphocyte subsets in peripheral blood of sheep vaccinated against D. nodosus. We also compare the use of melatonin in implants and in injections. Melatonin administration either as implants or by injection produced higher antibody titers against A1 and C serotypes compared to those animals that received only the vaccine. These results support the use of melatonin as an adjuvant in vaccination against D. nodosus. Firstly, melatonin induces higher antibody titer than the vaccine alone, secondly, melatonin increase IgG+ B lymphocytes and CD4+ T lymphocytes in vaccinated sheep. These results suggest that melatonin enhances T CD4 cell activation and subsequently secondary humoral immune responses. Further studies are required to determine the mechanism underlining the immunomodulatory role of melatonin in the context of vaccination.

B cell responses to apoptotic cells in MFG-E8-/- mice

Defective clearance of apoptotic cells in MFG-E8 deficient mice results in lupus-like disease in the mixed B6x129, but not pure B6 background. The lack of overt autoimmunity in MFG-E8-/- B6 mice suggests that accumulation of apoptotic cells is not sufficient to break central tolerance. However, the delayed clearance of apoptotic cells in the follicles of MFG-E8-/- B6 mice provides an excellent opportunity to investigate how B cells respond to excessive apoptotic cells in the periphery under relatively non-inflammatory conditions. In MFG-E8-/- B6 mice, we found increased IgG2c production against apoptotic cells and oxidized LDL. Apoptotic cell induced antibody responses depended on MyD88 signal and T cell help. In addition, MFG-E8-/- B6 mice had enlarged MZ B cell compartments as well as an enhanced antibody response to NP-Ficoll. Moreover, a significant percentage of MZ B cells in aged MFG-E8-/- B6 mice migrated into follicles. Injecting apoptotic cells or oxidized LDL into wild type mice as well as physiological accumulation of LDL in ApoE-/- mice recapitulated the translocation of MZ B cells. To determine how MFG-E8 deficiency affects the functions of autoreactive B cells specific for nucleic acids in the periphery under non-inflammatory conditions, we utilized BCR transgenic mice to bypass central selection and compared the differentiation of TLR9 dependent anti-dsDNA 56R B cells and TLR7 dependent anti-ssRNA H564 B cells in MFG-E8-/- mice. In MFG-E8-/- 56R mice, anti-dsDNA specific 56R/Vκ38c B cells differentiated into MZ B cells but not AFCs. On the contrary, in MFG-E8-/-H564 mice, anti-ssRNA specific H564 B cells further differentiated into GC B cells and AFCs. Adoptive transfer of activated autoreactive B cells confirmed that H564 B cells were more sensitive to apoptotic cell antigens than 56R B cells. Our observations provide new insights about the MZ B cell translocation in lupus patients as well as the dichotomy of TLR9 and TLR7 signals in the pathogenesis of lupus.

Co-delivery of a CD4 T cell helper epitope via covalent liposome attachment with a surface-arrayed B cell target antigen fosters higher affinity antibody responses

Liposomal vaccines incorporating adjuvant and CD4 T cell helper peptides enhance antibody responses against weakly immunogenic B cell epitopes such as found in the membrane proximal external region (MPER) of the HIV-1 gp41 subunit. While the inclusion of exogenous helper peptides in vaccine formulations facilitates stronger and more durable antibody responses, the helper peptide incorporation strategy per se may influence the overall magnitude and quality of B cell target antigen immunogenicity. Both variability in individual peptide encapsulation as well as the potential for liposome surface-associated helper peptides to misdirect the humoral response are potential parameters impacting outcome. In this study, we used MPER/liposome vaccines as a model system to examine how the mode of the potent LACK T helper peptide formulation modulates antibody responses against the MPER antigen. We directly compared liposome surface-arrayed palmitoyl LACK (pLACK) versus soluble LACK (sLACK) encapsulated in the liposomes and free in solution. Independent of LACK formulation methods, dendritic cell activation and LACK presentation were equivalent in vivo. The frequency of MPER-specific GC B cells promoted by sLACK was higher than that stimulated by pLACK formulation, a finding associated with a significantly greater frequency of LACK-specific GC B cells induced by pLACK. While there were no significant differences in the quantity of MPER-specific serological responses, the MPER-specific antibody titer trended higher with sLACK formulated vaccines at the lower dose of LACK. However, pLACK generated relatively greater MPER-specific antibody affinities than those induced by sLACK-formulated vaccines. Overall, the results suggest that liposomal surface-associated LACK enhances immunogenicity of LACK through better engagement of LACK-specific B cells. Of note, this is not detrimental to the induction of MPER-specific immune responses; rather, the elicitation of higher affinity anti-MPER antibodies benefits from augmented help delivered via covalent linkage of the pLACK CD4 T cell epitope in conjunction with MPER/liposome presentation.

Dyslipidemia promotes germinal center reactions via IL-27

Cardiovascular disease such as atherosclerosis is caused by imbalanced lipid metabolism and represents a leading cause of death worldwide. Epidemiological studies show that patients with systemic autoimmune diseases exhibit a higher incidence of atherosclerosis. Conversely, hyperlipidemia has been known to accelerate the incidence of autoimmune diseases in humans and in animal models. However, there is a considerable gap in our understanding of how atherosclerosis impacts the development of the autoimmunity in humans, and vice versa. The atherosclerosis-related autoimmune diseases include psoriasis, rheumatoid arthritis, systemic lupus erythematosus (SLE) and diabetes mellitus. By using animal models of atherosclerosis and SLE, we have recently demonstrated that hyperlipidemia significantly accelerates the development of autoantibodies, by inducing autoimmune follicular helper T (TFH) cells. Mechanistic studies have identified that hyperlipidemia induces IL-27 production in a TLR4-dependent manner, likely via downregulating LXR expression in dendritic cells. In this case, mice lacking IL-27 do not develop enhanced antibody responses. Thus it is noted that these findings propose a mechanistic insight responsible for the tight association between cardiovascular diseases and SLE in humans.

Impact of dietary spray-dried bovine plasma addition on pigs infected with porcine epidemic diarrhea virus.

Experimental data suggest that the addition of spray-dried plasma (SDP) to pig feed may enhance antibody responses against certain pathogens and negatively impact virus survival. The benefit of SDP on Escherichia coli infection is well documented. The aim of this study was to determine the effect of bovine SDP (BovSDP) in the pig diet on acute porcine epidemic diarrhea virus (PEDV) infection. A total of 16 3-wk-old conventional crossbred pigs were used and divided into three groups. Treatments included 1) a negative control group fed a commercial diet and sham inoculated with commercial liquid porcine plasma (n = 3), 2) a positive control group fed a commercial diet and inoculated with PEDV-spiked porcine plasma (PEDV; n = 8), and 3) a third group of pigs fed the commercial diet with inclusion of 5% spray-dried bovine plasma and inoculated with PEDV-spiked porcine plasma (BovSDP; n = 5). Although clinical signs associated with PEDV infection were mild in the BovSDP group, two of eight pigs in the PEDV group developed moderate clinical disease and had to be euthanized. The PEDV IgG and IgA antibody levels and prevalence rates were significantly (P < 0.05) higher in the PEDV–BovSDP group compared with the PEDV group at 7 d postinoculation. The average fecal PEDV RNA shedding time was 7.2 ± 1.0 d for the PEDV–BovSDP group and 9.3 ± 1.1 d for the PEDV group with an overall time to clearance of PEDV shedding of 11 d for PEDV–BovSDP pigs and at least 14 d for PEDV pigs, which was not different (P = 0.215). The results indicate that addition of BovSDP induced an earlier anti-PEDV antibody response in pigs experimentally infected with PEDV thereby reducing clinical disease and the amount and duration of viral shedding during acute PEDV infection.

αv Integrins regulate germinal center B cell responses through noncanonical autophagy.

Germinal centers (GCs) are major sites of clonal B cell expansion and generation of long-lived, high-affinity antibody responses to pathogens. Signaling through TLRs on B cells promotes many aspects of GC B cell responses, including affinity maturation, class switching, and differentiation into long-lived memory and plasma cells. A major challenge for effective vaccination is identifying strategies to specifically promote GC B cell responses. Here, we have identified a mechanism of regulation of GC B cell TLR signaling, mediated by αv integrins and noncanonical autophagy. Using B cell-specific αv-KO mice, we show that loss of αv-mediated TLR regulation increased GC B cell expansion, somatic hypermutation, class switching, and generation of long-lived plasma cells after immunization with virus-like particles (VLPs) or antigens associated with TLR ligand adjuvants. Furthermore, targeting αv-mediated regulation increased the magnitude and breadth of antibody responses to influenza virus vaccination. These data therefore identify a mechanism of regulation of GC B cells that can be targeted to enhance antibody responses to vaccination.

Removal of the N-Glycosylation Sequon at Position N116 Located in p27 of the Respiratory Syncytial Virus Fusion Protein Elicits Enhanced Antibody Responses after DNA Immunization.

Prevention of severe lower respiratory tract infections in infants caused by the human respiratory syncytial virus (hRSV) remains a major public health priority. Currently, the major focus of vaccine development relies on the RSV fusion (F) protein since it is the main target protein for neutralizing antibodies induced by natural infection. The protein conserves 5 N-glycosylation sites, two of which are located in the F2 subunit (N27 and N70), one in the F1 subunit (N500) and two in the p27 peptide (N116 and N126). To study the influence of the loss of one or more N-glycosylation sites on RSV F immunogenicity, BALB/c mice were immunized with plasmids encoding RSV F glycomutants. In comparison with F WT DNA immunized mice, higher neutralizing titres were observed following immunization with F N116Q. Moreover, RSV A2-K-line19F challenge of mice that had been immunized with mutant F N116Q DNA was associated with lower RSV RNA levels compared with those in challenged WT F DNA immunized animals. Since p27 is assumed to be post-translationally released after cleavage and thus not present on the mature RSV F protein, it remains to be elucidated how deletion of this glycan can contribute to enhanced antibody responses and protection upon challenge. These findings provide new insights to improve the immunogenicity of RSV F in potential vaccine candidates.

Sublingual administration of 2-hydroxyethyl methacrylate enhances antibody responses to co-administered ovalbumin and Streptococcus mutans

Objective: The oral mucosa of patients undergoing dental procedures is often exposed to residual monomers leaking from incompletely cured acrylic resins. We investigated whether 2-hydroxyethyl methacrylate (HEMA) monomers applied to the sublingual mucosa in mice modulate the antibody responses towards co-administered ovalbumin (OVA) or live oral bacteria.Material and methods: OVA, live mouse oral commensal Lactobacillus murinus or live human oral commensal Streptococcus mutans were administered sublingually with or without HEMA to BALB/c mice on four weekly occasions. One week after the last administration, the experiment was terminated and serum antibody levels were analyzed using ELISA.Results: Significantly increased IgG and IgE anti-OVA antibody activity was found in the sera from mice immunized with OVA together with HEMA, as compared to mice immunized with OVA alone. Likewise, S. mutans together with HEMA induced an IgG anti-S. mutans antibody response that was significantly higher than the antibody response detected after application of S. mutans alone. No IgG anti-L. murinus antibody response was detected in mice immunized with L. murinus together with HEMA, as compared to the background activity.Conclusions: We report that HEMA monomers have adjuvant properties when sublingually administered in combination with OVA or S. mutans.

Human Memory B Cells TargetingStaphylococcus aureusExotoxins Are Prevalent with Skin and Soft Tissue Infection

ABSTRACTStaphylococcus aureus is a Gram-positive opportunistic pathogen that causes superficial and invasive infections in the hospital and community. High mortality from infection emphasizes the need for improved methods for prevention and treatment. Although S. aureus possesses an arsenal of virulence factors that contribute to evasion of host defenses, few studies have examined long-term humoral and B-cell responses. Adults with acute-phase skin and soft tissue infections were recruited; blood samples were obtained; and S. aureus isolates, including methicillin-resistant strains, were subjected to genomic sequence analysis. In comparisons of acute-phase sera with convalescent-phase sera, a minority (37.5%) of patients displayed 2-fold or greater increases in antibody titers against three or more S. aureus antigens, whereas nearly half exhibited no changes, despite the presence of toxin genes in most infecting strains. Moreover, enhanced antibody responses waned over time, which could reflect a defect in B-cell memory or long-lived plasma cells. However, memory B cells reactive with a range of S. aureus antigens were prevalent at both acute-phase and convalescent-phase time points. While some memory B cells exhibited toxin-specific binding, those cross-reactive with structurally related leucocidin subunits were dominant across patients, suggesting the targeting of conserved epitopes. Memory B-cell reactivity correlated with serum antibody levels for selected S. aureus exotoxins, suggesting a relationship between the cellular and humoral compartments. Overall, although there was no global defect in the representation of anti-S. aureus memory B cells, there was evidence of restrictions in the range of epitopes recognized, which may suggest potential therapeutic approaches for augmenting host defenses.

Overcoming the Neonatal Limitations of Inducing Germinal Centers through Liposome-Based Adjuvants Including C-Type Lectin Agonists Trehalose Dibehenate or Curdlan

Neonates and infants are more vulnerable to infections and show reduced responses to vaccination. Consequently, repeated immunizations are required to induce protection and early life vaccines against major pathogens such as influenza are yet unavailable. Formulating antigens with potent adjuvants, including immunostimulators and delivery systems, is a demonstrated approach to enhance vaccine efficacy. Yet, adjuvants effective in adults may not meet the specific requirements for activating the early life immune system. Here, we assessed the neonatal adjuvanticity of three novel adjuvants including TLR4 (glucopyranosyl lipid adjuvant-squalene emulsion), TLR9 (IC31®), and Mincle (CAF01) agonists, which all induce germinal centers (GCs) and potent antibody responses to influenza hemagglutinin (HA) in adult mice. In neonates, a single dose of HA formulated into each adjuvant induced T follicular helper (TFH) cells. However, only HA/CAF01 elicited significantly higher and sustained antibody responses, engaging neonatal B cells to differentiate into GCs already after a single dose. Although antibody titers remained lower than in adults, HA-specific responses induced by a single neonatal dose of HA/CAF01 were sufficient to confer protection against influenza viral challenge. Postulating that the neonatal adjuvanticity of CAF01 may result from the functionality of the C-type lectin receptor (CLR) Mincle in early life we asked whether other C-type lectin agonists would show a similar neonatal adjuvanticity. Replacing the Mincle agonist trehalose 6,6′-dibehenate by Curdlan, which binds to Dectin-1, enhanced antibody responses through the induction of similar levels of TFH, GCs and bone marrow high-affinity plasma cells. Thus, specific requirements of early life B cells may already be met after a single vaccine dose using CLR-activating agonists, identified here as promising B cell immunostimulators for early life vaccines when included into cationic liposomes.

Stinging nettle and neem enhance antibody response to local killed and imported live infectious bursal disease vaccines in indigenous chicken in Kenya

Immune responses are critical for protection of chickens from infectious bursal disease (IBD). In this study, the antibody response-enhancing effect of drinking water supplementation of 1% stinging nettle and neem on different IBD vaccines and vaccination regimes was evaluated, using 36 (n = 36) specific antibody negative indigenous chicks. The birds were allocated into 3 groups as follows: 1A-C, 2A-C, and 3A-B, while group 3C acted as the unvaccinated non-supplemented control. A local inactivated K1 and imported live attenuated D78 IBD vaccines were given to groups 1A-C and 3A-B at 14 and 28 d of age, respectively. A combination of K1 and D78 vaccines was given 30 d apart to groups 2A and 2B (D78 at 14 and 21 d and K1 at 44 d of age) and on the same d to group 2C at 14 and 28 d of age. Stinging nettle was given in water to groups 1B, 2B, and 2C, and neem to groups 1C, 2A, and 3B. Birds were bled weekly and immune responses monitored using indirect ELISA. Both neem and stinging nettle had antibody response-enhancing effects in groups 1B and 1C, receiving the local inactivated K1 vaccine. There were significant differences (P < 0.05) in antibody titers between groups 1A and 2C. Stinging nettle induced earlier onset of high antibody responses in group 2C and persistent titers (>3.8 log10) from the third week in group 2B. Imported live D78 vaccine induced higher antibody titers compared to the local inactivated K1 vaccine. Groups 2B and 2C receiving a combination of the local K1 and imported live attenuated D78 vaccines had the highest antibody titers. Adoption of stinging nettle supplementation and a prime-boost program involving use of a local virus isolates-derived vaccine is recommended.

Enhanced Antibody Responses in a Novel NOG Transgenic Mouse with Restored Lymph Node Organogenesis.

Lymph nodes (LNs) are at the center of adaptive immune responses. Various exogenous substances are transported into LNs and a series of immune responses ensue after recognition by antigen–specific lymphocytes. Although humanized mice have been used to reconstitute the human immune system, most lack LNs due to deficiency of the interleukin (IL)-2Rγ gene (cytokine common γ chain, γc). In this study, we established a transgenic strain, NOG-pRORγt-γc, in the NOD/shi-scid-IL-2Rγnull (NOG) background, in which the γc gene was expressed in a lymph-tissue inducer (LTi) lineage by the endogenous promoter of RORγt. In this strain, LN organogenesis was normalized and the number of human T cells substantially increased in the periphery after reconstitution of the human immune system by human hematopoietic stem cell transplantation. The distribution of human T cells differed between NOG-pRORγt-γc Tg and NOG-non Tg mice. About 40% of human T cells resided in LNs, primarily the mesenteric LNs. The LN-complemented humanized mice exhibited antigen-specific immunoglobulin G responses together and an increased number of IL-21+–producing CD4+ T cells in LNs. This novel mouse strain will facilitate recapitulation of human immune responses.

Voluntary Wheel Running Does Not Alter Mortality to or Immunogenicity of Vaccinia Virus in Mice: A Pilot Study.

Exercise has been shown to improve immune responses to viral infections and vaccines in several mouse models. However, previous pathogen studies have primarily used infections limited to the respiratory tract. Additionally, previous studies have utilized forced treadmill exercise paradigms, and voluntary wheel running (VWR) has been shown to have differential effects on the immune system in non-infection models. We examined whether VWR could improve morbidity and mortality to a 50% lethal dose of vaccinia virus (VACV), a systemic pathogen commonly used to examine immune responses. Additionally, we examined whether VWR could improve antibody response to a replication-deficient strain of VACV, mimicking a vaccination. Male C57Bl/6J mice underwent 8 weeks of VWR or remained sedentary, then were infected intranasally with 105 PFU VACV strain WR and followed 14 days for weight loss. Mice in the vaccination study ran or were sedentary for 8 weeks, then were given 106 PFU of replication-deficient VACV strain MVA intraperitoneally. Blood was collected at 1, 2, and 4 weeks post-inoculation, and anti-VACV IgG titer was determined by ELISA. VWR did not improve mortality due to VACV infection (p = 0.26), although fewer VWR mice (4/10) died compared to sedentary (SED, 6/10). VWR did not prevent body weight loss due to infection compared to SED (p = 0.20), although VWR mice loss slightly less weight compared to SED through the first 6 days post-infection. Food intake was significantly reduced in SED post-infection compared to VWR (p = 0.05). VWR mice developed a greater IgG antibody response, although this was not significant (p = 0.22). In summary, VWR did not protect against mortality to VACV or prevent infection-induced weight loss, and VWR did not enhance antibody responses. However, there were non-significant trends toward VWR-related improvements in these outcomes, and post-infection food intake was improved by VWR.

Antibody responses among adolescent females receiving two or three quadrivalent human papillomavirus vaccine doses at standard and prolonged intervals

BackgroundThe originally recommended dosing schedule, 0, 2, 6 months, for the 3-dose quadrivalent human papillomavirus vaccine (4vHPV) was often not followed, resulting in longer than recommended intervals between doses and interest in the effect of prolonged intervals. Recent two-dose recommendations require investigations into the effect of delaying dose 2. MethodsThis multi-site, prospective study enrolled healthy 9–17 year old girls (n = 1321) on the day of or within 28 days following a third dose of 4vHPV vaccination. Antibody titers to 4vHPV types were measured at one and six months post-dose 3 from all participants and post-dose 2 from participants who were on time for dose 3. To compare antibody responses, participants were categorized into groups: second and third doses on time (control group); on-time dose 2, substantially late dose 3 (group 2); substantially late dose 2, on-time dose 3 (group 3); both doses substantially late (group 4). Analyses compared age-adjusted geometric mean titers (GMTs) at one-month and six-months post-dose 3, effect of delaying the second dose, and two versus three doses as well as post-dose 2 GMTs, stratified by age. ResultsCompared to on-time dosing, one-month post-dose 3 GMTs were non-inferior in groups 2, 3, and 4 and were superior in group 2. Six month post-dose 3 GMTs were superior in groups 2, 3, and 4 for each genotype, except HPV 18 in group 3. Age-adjusted post does 2 titers were significantly lower than post-dose 3 titers when dose 2 was on time but were significantly higher when dose 2 was substantially late. Participants ≥15 years old had no difference in post-dose 2 titers compared to <15 year olds when dose 2 was substantially delayed. ConclusionsProlonged intervals between doses do not appear to diminish and may enhance antibody response to 4vHPV.ClinicalTrials.gov (NCT00524745).

Phenotypic and genotypic analyses of an attenuated porcine reproductive and respiratory syndrome virus strain after serial passages in cultured porcine alveolar macrophages.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine viral pathogen that causes major economic losses worldwide. We previously reported an over-attenuated phenotype of cell-adapted PRRSV strain CA-2-P100 in vivo. In the present study, CA-2-P100 was serially propagated in cultured porcine alveolar macrophage (PAM) cells for up to 20 passages to obtain the derivative strain CA-2-MP120. Animal inoculation studies revealed that both CA-2-P100 and CA-2-MP120 had decreased virulence, eliciting weight gains, body temperatures, and histopathologic lesions similar to those in the negative control group. However, compared to CA-2-P100 infection, CA-2-MP120 yielded consistently higher viremia kinetics and enhanced antibody responses in pigs. All pigs inoculated with CA-2-MP120 developed viremia and seroconverted to PRRSV. During 20 passages in PAM cells, CA-2-MP120 acquired 15 amino acid changes that were mostly distributed in nsp2 and minor structural protein-coding regions. Among these changes, 6 mutations represented reversions to the sequences of the reference CA-2 and parental CA-2-P20 strains. These genetic drifts may be hypothetical molecular markers associated with PRRSV macrophage tropism and virulence. Our results indicate that the PAM-passaged CA-2-MP120 strain is a potential candidate for developing a live, attenuated PRRSV vaccine.

PI3K- and PTEN-Governed PIP&lt;sub&gt;2&lt;/sub&gt; and PIP&lt;sub&gt;3&lt;/sub&gt; Equilibrium Regulates the Activation and Function of B Cells Through Dock2 Controlled Growth of Antigen Receptor Microclusters

The growth of B cell receptor (BCR) microclusters upon antigen stimulation is one of the most important events in B cell activation, however the underlying molecular mechanism unknown. Here, we systematically addressed this question through a combination of molecular imaging, genetic ablation, pharmacological inhibitor and mice model approaches. We showed that the earliest BCR signaling molecules, Lyn, Syk and PLCγ2 are required for B cells to initiate the growth of BCR microclusters. PI3K-mediated PIP3 production but not Akt and PDK1 activation was required for the sustained growth of the BCR microclusters. The PI3K counterpart signaling molecule, PTEN, potently inhibited the growth of the BCR microclusters, which was dependent on its membrane binding activities and on the lipid phosphatase but not protein phosphatase activity. Mechanistically, we showed that PIP3-dependent recruitment and activation of a unique guanine nucleotide exchange factor, Dock2, was required for the sustained growth of the BCR microclusters through the remodeling of the F-actin cytoskeleton. As a consequence, the formation of the antigen-induced B cell immunological synapse (IS) in Dock2 deficient B cells was drastically impaired. Moreover, Dock2 deficient B cells exhibited decreased Ca2 influx. Physiologically, we found that mice with reduced expression of PTEN in B cells showed enhanced antibody responses and a spontaneous lupus-like autoimmune phenotype. Clinically, we showed that primary B cells from lupus patients exhibited more prominent BCR and PI3K microclusters than B cells from healthy controls. Thus, these results demonstrated the importance of PI3K- and PTEN-governed PIP2 and PIP3 equilibrium in regulating the dynamic growth of BCR microclusters during the initiation of B cell activation.

HIV-1 gp120 and Modified Vaccinia Virus Ankara (MVA) gp140 Boost Immunogens Increase Immunogenicity of a DNA/MVA HIV-1 Vaccine.

ABSTRACTAn important goal of human immunodeficiency virus (HIV) vaccine design is identification of strategies that elicit effective antiviral humoral immunity. One novel approach comprises priming with DNA and boosting with modified vaccinia virus Ankara (MVA) expressing HIV-1 Env on virus-like particles. In this study, we evaluated whether the addition of a gp120 protein in alum or MVA-expressed secreted gp140 (MVAgp140) could improve immunogenicity of a DNA prime-MVA boost vaccine. Five rhesus macaques per group received two DNA primes at weeks 0 and 8 followed by three MVA boosts (with or without additional protein or MVAgp140) at weeks 18, 26, and 40. Both boost immunogens enhanced the breadth of HIV-1 gp120 and V1V2 responses, antibody-dependent cellular cytotoxicity (ADCC), and low-titer tier 1B and tier 2 neutralizing antibody responses. However, there were differences in antibody kinetics, linear epitope specificity, and CD4 T cell responses between the groups. The gp120 protein boost elicited earlier and higher peak responses, whereas the MVAgp140 boost resulted in improved antibody durability and comparable peak responses after the final immunization. Linear V3 specific IgG responses were particularly enhanced by the gp120 boost, whereas the MVAgp140 boost also enhanced responses to linear C5 and C2.2 epitopes. Interestingly, gp120, but not the MVAgp140 boost, increased peak CD4+ T cell responses. Thus, both gp120 and MVAgp140 can augment potential protection of a DNA/MVA vaccine by enhancing gp120 and V1/V2 antibody responses, whereas potential protection by gp120, but not MVAgp140 boosts, may be further impacted by increased CD4+ T cell responses.IMPORTANCE Prior immune correlate analyses with humans and nonhuman primates revealed the importance of antibody responses in preventing HIV-1 infection. A DNA prime-modified vaccinia virus Ankara (MVA) boost vaccine has proven to be potent in eliciting antibody responses. Here we explore the ability of boosts with recombinant gp120 protein or MVA-expressed gp140 to enhance antibody responses elicited by the GOVX-B11 DNA prime-MVA boost vaccine. We found that both types of immunogen boosts enhanced potentially protective antibody responses, whereas the gp120 protein boosts also increased CD4+ T cell responses. Our data provide important information for HIV vaccine designs that aim for effective and balanced humoral and T cell responses.

Minimal Sex-Differential Modulation of Reactivity to Pathogens and Toll-Like Receptor Ligands following Infant Bacillus Calmette-Guérin Russia Vaccination.

Bacillus Calmette–Guérin (BCG), the only licensed vaccine against tuberculosis, has been shown to provide heterologous protection against unrelated pathogens and enhance antibody responses to several routine expanded program on immunization (EPI) vaccines. Understanding these heterologous effects is important for the development of optimal vaccination strategies. We set out to assess the effect of vaccination with BCG Russia of 6-week-old infants on in vitro reactivity to a panel of toll-like receptor (TLR) agonists (TLR2, 4, and 7/8) and heat-killed pathogens [Streptococcus pneumoniae, Candida albicans (CA), and Escherichia coli], and antibody responses to other EPI vaccines compared to BCG naïve infants. We observed no effect of BCG vaccination on innate (TNF-α) or Th2 (IL-4) cytokine responses, but found enhanced CA-specific CD8+IFN-γ+ responses in BCG vaccinated males and females 1 week after vaccination and decreased IFN-γ:IL4 ratio to SP in females. By 12 weeks (but not 1 week) of post-vaccination, there was significant downmodulation of Th1 cytokine responses in BCG vaccinated infants; and TLR-stimulated IL-10 and IL-17 responses declined in BCG vaccinated females but not males. Significant changes also occurred in the BCG naïve group, mainly at 18 weeks, including decreased Th1 and increased IL-10 responses. The effects at 18 weeks were most likely a result of immune modulation by the intervening EPI vaccines given at 8, 12, and 16 weeks of age. There was no effect of BCG vaccination on EPI antibody levels at either time point. Taken together, our results support minimal early heterologous immune modulation by BCG Russia vaccination that did not persist 12 weeks after vaccination.

IgG3-antigen complexes are deposited on follicular dendritic cells in the presence of C1q and C3

IgG3, passively administered together with small proteins, induces enhanced primary humoral responses against these proteins. We previously found that, within 2 h of immunization, marginal zone (MZ) B cells capture IgG3-antigen complexes and transport them into splenic follicles and that this requires the presence of complement receptors 1 and 2. We have here investigated the localization of IgG3 anti-2, 4, 6-trinitrophenyl (TNP)/biotin-ovalbumin-TNP immune complexes in the follicles and the involvement of classical versus total complement activation in this process. The majority (50–90%) of antigen inside the follicles of mice immunized with IgG3-antigen complexes co-localized with the follicular dendritic cell (FDC) network. Capture of antigen by MZ B cells as well as antigen deposition on FDC was severely impaired in mice lacking C1q or C3, and lack of either C1q or C3 also impaired the ability of IgG3 to enhance antibody responses. Finally, IgG3 efficiently primed for a memory response against small proteins as well as against the large protein keyhole limpet hemocyanine.

Identification of Biologically Active Pyrimido[5,4-b]indoles That Prolong NF-κB Activation without Intrinsic Activity.

Most vaccine adjuvants directly stimulate and activate antigen presenting cells but do not sustain immunostimulation of these cells. A high throughput screening (HTS) strategy was designed to identify compounds that would sustain NF-κB activation by a stimulus from the Toll-like receptor (TLR)4 ligand, lipopolysaccharide (LPS). Several pilot studies optimized the parameters and conditions for a cell based NF-κB reporter assay in human monocytic THP-1 cells. The final assay evaluated prolongation of LPS induced NF-κB activation at 12 h. The dynamic range of the assay was confirmed in a pilot screen of 14 631 compounds and subsequently in a main extensive screen with 166 304 compounds. Hit compounds were identified using an enrichment strategy based on unsupervised chemoinformatic clustering, and also by a naı̈ve "Top X" approach. A total of 2011 compounds were then rescreened for levels of coactivation with LPS at 5 h and 12 h, which provided kinetic profiles. Of the 407 confirmed hits, compounds that showed correlation of the kinetic profiles with the structural similarities led to identification of four chemotypes: pyrimido[5,4-b]indoles, 4H-chromene-3-carbonitriles, benzo[d][1,3]dioxol-2-ylureas, and tetrahydrothieno[2,3-c]pyridines, which were segregated by 5 h and 12 h kinetic characteristics. Unlike the TLR4 agonistic pyrimidoindoles identified in previous studies, the revealed pyrimidoindoles in the present work did not intrinsically stimulate TLR4 nor induce NF-κB but rather prolonged NF-κB signaling induced by LPS. A 42-member combinatorial library was synthesized which led to identification of potent N3-alkyl substituted pyrimidoindoles that were not only active in vitro but also enhanced antibody responses in vivo when used as a coadjuvant. The novel HTS strategy led to identification of compounds that are intrinsically quiescent but functionally prolong stimulation by a TLR4 ligand and thereby potentiate vaccine efficacy.