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Abstract Background The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Nef, is an attractive vaccine target because it is involved in viral pathogenesis, is expressed early in the viral life cycle and harbors many T and B cell epitopes. Several clinical trials include gene-based vaccines encoding this protein. However, Nef has been shown to transform certain cell types in vitro. Based on these findings we performed a long-term toxicity and immunogenicity study of Nef, encoded either by Modified Vaccinia virus Ankara or by plasmid DNA. BALB/c mice were primed twice with either DNA or MVA encoding Nef and received a homologous or heterologous boost ten months later. In the meantime, the Nef-specific immune responses were monitored and at the time of sacrifice an extensive toxicological evaluation was performed, where presence of tumors and other pathological changes were assessed. Results The toxicological evaluation showed that immunization with MVAnef is safe and does not cause cellular transformation or other toxicity in somatic organs. Both DNAnef and MVAnef immunized animals developed potent Nef-specific cellular responses that declined to undetectable levels over time, and could readily be boosted after almost one year. This is of particular interest since it shows that plasmid DNA vaccine can also be used as a potent late booster of primed immune responses. We observed qualitative differences between the T cell responses induced by the two different vectors: DNA-encoded nef induced long-lasting CD8+ T cell memory responses, whereas MVA-encoded nef induced CD4+ T cell memory responses. In terms of the humoral immune responses, we show that two injections of MVAnef induce significant anti-Nef titers, while repeated injections of DNAnef do not. A single boost with MVAnef could enhance the antibody response following DNAnef prime to the same level as that observed in animals immunized repeatedly with MVAnef. We also demonstrate the possibility to boost HIV-1 Nef-specific immune responses using the MVAnef construct despite the presence of potent anti-vector immunity. Conclusion This study shows that the nef gene vectored by MVA does not induce malignancies or other adverse effects in mice. Further, we show that when the nef gene is delivered by plasmid or by a viral vector, it elicits potent and long-lasting immune responses and that these responses can be directed towards a CD4+ or a CD8+ T cell response depending on the choice of vector.

Abstract New potent vaccine adjuvants are desirable for increasing the efficacy of novel vaccine modalities such as DNA and peptides. We therefore tested if syngeneic erythrocytes could serve as delivery vectors for selected HIV peptides and compared the potency of these constructs to immunization with peptides in phosphate buffered saline or in incomplete Freunds adjuvant. Immunization of mice with peptides in a low dose (5 ng) coupled to erythrocytes induced a weak immune response in mice. These peptides alone (5 μg) gave no immune responses, while formulating the peptides (50 μg) in IFA induced strong homologous immunity as well as prominent cross reactivity to a related mutant epitope. Thus, vaccine delivery using syngeneic erythrocytes, although attractive for clinical use, might be of limited value due to the low amount of antigen that can be loaded per erythrocyte.

We aimed to use the digital platform maintained by the local health service providers in Southeast Sweden for integrated monitoring of disparities in vaccination and morbidity during the COVID-19 pandemic. The monitoring was performed in the adult population of two counties ( n = 657,926) between 1 February 2020 and 15 February 2022. The disparities monitored were relocated (internationally displaced), substance users, and suffering from a psychotic disorder. The outcomes monitored were COVID-19 vaccination, SARS-CoV-2 test results, and hospitalization with COVID-19. Relocated residents displayed an increased likelihood of remaining unvaccinated and a decreased likelihood of testing as well as increased risks of primary SARS-CoV-2 infection and hospitalization compared with the general population. Suffering from a major psychiatric disease was associated with an increased risk of remaining unvaccinated and an increased risk of hospitalization but a decreased risk of SARS-CoV-2 infection. From the digital monitoring, we concluded that the relocated minority received insufficient protection during the pandemic, suggesting the necessity for comprehensive promotion of overall social integration. Persons with major psychiatric diseases underused vaccination, while they benefitted from proactively provided testing, implying a need for active encouragement of vaccination. Further research is warranted on legal and ethical frameworks for digital monitoring in vaccination programs.

Currently, vaccine development against different respiratory diseases is at its peak. It is of utmost importance to find suitajble adjuvants that can increase the potency of the vaccine candidates. This study aimed to determine the systemic and splenic immune mechanisms in mice models induced by anionic and cationic lipid adjuvants in the presence of the vaccine-candidate influenza antigen hemagglutinin (HA). In the presence of the HA antigen, the cationic adjuvant (N3) increased conventional dendritic cell 1 (cDC1) abundance with enhanced MHCI and CD80-CD86 costimulatory marker expression, and significantly higher CD8T and Th17 populations with enhanced interferon-gamma (IFNγ) expression in CD8T and CD4T populations. Conversely, the anionic adjuvant (L3) increased the cDC2 population percentage with significantly higher MHCII and DEC205 expression, along with an increase in the CD4T and regulatory T cell populations. The L3-treated group also exhibited higher percentages of activated B and plasma cell populations with significantly higher antigen-specific IgG and IgA titer and virus neutralization potential. While the anionic adjuvant induced significantly higher humoral responses than the cationic adjuvant, the latter influenced a significantly higher Th1/Th17 response. For customized vaccine development, it is beneficial to have alternative adjuvants that can generate differential immune responses with the same vaccine candidate antigen. This study will aid the selection of adjuvants based on their charges to improve specific immune response arms in the future development of vaccine formulation.

Immunization against influenza through vaccination is the most effective method with which to prevent infection. To assess protection after immunization, analysing humoral response with a hemagglutinin inhibition assay is the gold standard, but cell-mediated immune response has been shown to better correlate with protection in the elderly. Our aim was to explore the influenza-specific cell-mediated and mucosal humoral responses in serologically defined responders and non-responders. We analysed sera for total immunoglobulins (Ig) A, G, and M and nasal swab samples for influenza-specific IgA. Peripheral blood mononuclear cells were stimulated with trivalent influenza vaccine VaxiGripTetra, and supernatants were analysed for influenza-specific responses with the Olink Immune-Oncology panel using a proximity extension assay. We included 73 individuals, of which 69 completed the study with follow-up sampling at one and six months post-vaccination. Of the 73, 51 (70%) were found to be serological responders and 22 (30%) were non-responders. We did not find any significant differences in sex or mucosal humoral response between responders and non-responders; however, a higher IFNγ/IL-10 ratio in individuals ≤65 years of age indicates an enhanced cell-mediated immune response in this age group. Characteristics of the non-responders were found to be higher levels of IgM, Granzyme B and Interleukin 12, and lower levels of C-X-C motif chemokine 13 compared with those of the responders. In conclusion, our results did not show any correlation between serological response and age. Furthermore, the majority of influenza-specific cell-mediated immune markers did not differ between responders and non-responders; the immune marker profile of the non-responders and its contribution to protection is of interest but needs to be further explored.

Seasonal influenza vaccination has different implications on the immune response depending on the comorbidities. Diabetes is one such critical disease that increases the patient's susceptibility to influenza and suppresses vaccine efficacy and immunity. The sex of the individuals also plays a definitive role in the immune responses to both the vaccine and the infection. This study aims to understand the efficacy of the seasonal vaccine against influenza in diabetic groups and undergoing immune mechanisms in different sexes (females and males). In this study, we are reporting about a switching of the immune response of the infected and vaccinated diabetic females towards stronger Th1/Th17 responses with suppressed humoral immunity. They show increased cDC1, enhanced proinflammatory activities within T cells, CD8T activation, Th17 proliferation, and the majority of IgG2 antibody subtypes with reduced neutralization potential. Males with diabetes exhibit enhanced humoral Th2-immunity than the nondiabetic group. They exhibit higher cDC2, and DEC205 levels within them with an increase in plasma B lymphocytes, higher IgG1 subtypes in plasma cells, and influenza-hemagglutinin-specific IgG titer with stronger virus neutralization potential. Males with diabetes recovered better than the females as observed from the changes in their body weight. This study highlights the critical immune mechanisms and sex-specific swapping of their preferred immune response pathways against influenza after vaccination during diabetes. We propose a need for a sex-specific customized vaccine regimen to be implemented against influenza for individuals having diabetes to exploit the manifested strength and weakness in their protective immunity., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Anirban Sengupta et al.)

Despite the enormous advancements in nanomedicine research, a limited number of nanoformulations are available on the market, and few have been translated to clinics. An easily scalable, sustainable, and cost-effective manufacturing strategy and long-term stability for storage are crucial for successful translation. Here, we report a system and method to instantly formulate NF achieved with a nanoscale polyelectrolyte coacervate-like system, consisting of anionic pseudopeptide poly(l-lysine isophthalamide) derivatives, polyethylenimine, and doxorubicin (Dox) via simple "mix-and-go" addition of precursor solutions in seconds. The coacervate-like nanosystem shows enhanced intracellular delivery of Dox to patient-derived multidrug-resistant (MDR) cells in 3D tumor spheroids. The results demonstrate the feasibility of an instant drug formulation using a coacervate-like nanosystem. We envisage that this technique can be widely utilized in the nanomedicine field to bypass the special requirement of large-scale production and elongated shelf life of nanomaterials.

Potent broad-spectrum antiviral agents are urgently needed to combat existing and emerging viral infections. This is particularly important considering that vaccine development is a costly and time consuming process and that viruses constantly mutate and render the vaccine ineffective. Antimicrobial peptides (AMP), such as bacteriocins, are attractive candidates as antiviral agents against enveloped viruses. One of these bacteriocins is PLNC8 αβ, which consists of amphipathic peptides with positive net charges that display high affinity for negatively charged pathogen membrane structures, including phosphatidylserine rich lipid membranes of viral envelopes. Due to the morphological and physiological differences between viral envelopes and host cell plasma membranes, PLNC8 αβ is thought to have high safety profile by specifically targeting viral envelopes without effecting host cell membranes. In this study, we have tested the antiviral effects of PLNC8 αβ against the flaviviruses Langat and Kunjin, coronavirus SARS-CoV-2, influenza A virus (IAV), and human immunodeficiency virus-1 (HIV-1). The concentration of PLNC8 αβ that is required to eliminate all the infective virus particles is in the range of nanomolar (nM) to micromolar (μM), which is surprisingly efficient considering the high content of cholesterol (8-35%) in their lipid envelopes. We found that viruses replicating in the endoplasmic reticulum (ER)/Golgi complex, e.g. SARS-CoV-2 and flaviviruses, are considerably more susceptible to PLNC8 αβ, compared to viruses that acquire their lipid envelope from the plasma membrane, such as IAV and HIV-1. Development of novel broad-spectrum antiviral agents can significantly benefit human health by rapidly and efficiently eliminating infectious virions and thereby limit virus dissemination and spreading between individuals. PLNC8 αβ can potentially be developed into an effective and safe antiviral agent that targets the lipid compartments of viral envelopes of extracellular virions, more or less independent of virus antigenic mutations, which faces many antiviral drugs and vaccines., Competing Interests: A patent application has been submitted to the Swedish Patent and Registration Office by Hazem Khalaf, Torbjörn Bengtsson, Jorma Hinkula, Wessam Melik, Daniel Aili, and Robert Selegård. The authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2022 Omer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Treatment with RNAi against HIV-1 transcripts efficiently inhibits viral replication but induces selection of escape mutants; therefore, the CCR5 coreceptor was suggested as an additional target. Blocking viral and host transcripts improved the antiviral effect. We have used short hairpin RNA (shRNA) targeting the human CCR5 (shCCR5) or the HIV-1 rev (shRev) transcripts to demonstrate distinctive properties of anti-CCR5 shRNA: shCCR5 induced more sustained protection than shRev; partial reduction in CCR5 expression substantially decreased HIV-1 infection, and shCCR5 performed better than shRev in the mixed shRNA-treated and untreated cultures. These observations indicate that CCR5 inhibitors should be conveniently included in HIV-1 gene silencing treatment schedules when only a certain cell fraction is protected to further reduce endogenous virus in a properly ART-treated HIV-1 infected individual., (© 2022. The Author(s).)

Despite the great success of vaccines over two centuries, the conventional strategy is based on attenuated/altered microorganisms. However, this is not effective for all microbes and often fails to elicit a protective immune response, and sometimes poses unexpected safety risks. The expanding nano toolbox may overcome some of the roadblocks in vaccine development given the plethora of unique nanoparticle (NP)-based platforms that can successfully induce specific immune responses leading to exciting and novel solutions. Nanovaccines necessitate a thorough understanding of the immunostimulatory effect of these nanotools. We present a comprehensive description of strategies in which nanotools have been used to elicit an immune response and provide a perspective on how nanotechnology can lead to future personalized nanovaccines., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The severity of the coronavirus disease in 2019 (COVID-19) is strongly linked to a dysregulated immune response. This fuels the fear of severe disease in patients with autoimmune disorders continuously using immunosuppressive/immunomodulating medications. One complication of COVID-19 is thromboembolism caused by intravascular aggregates of neutrophil extracellular traps (NETs) occluding the affected vessels. Like COVID-19, systemic lupus erythematosus (SLE) is characterized by, amongst others, an increased risk of thromboembolism. An imbalance between NET formation and clearance is suggested to play a prominent role in exacerbating autoimmunity and disease severity. Serologic evidence of exposure to SARS-CoV-2 has a minor impact on the SLE course in a Swedish cohort reportedly. Herein, we assessed NET formation in patients from this cohort by neutrophil elastase (NE) activity and the presence of cell-free DNA, MPO-DNA, and NE-DNA complexes and correlated the findings to the clinical parameters. The presence of NE-DNA complexes and NE activity differed significantly in pre-pandemic versus pandemic serum samples. The latter correlated significantly with the hemoglobin concentration, blood cell counts, and complement protein 3 and 4 levels in the pre-pandemic but only with the leukocyte count and neutrophil levels in the pandemic serum samples. Taken together, our data suggest a change, especially in the NE activity independent of exposure to SARS-CoV-2.

The term hybrid immunity is used to denote the immunological status of vaccinated individuals with a history of natural infection. Reports of new SARS-CoV-2 variants of concern motivate continuous rethought and renewal of COVID-19 vaccination programs. We used a naturalistic case-control study design to compare the effectiveness of the BNT162b2 mRNA vaccine to hybrid immunity 180 days post-vaccination in prioritized and non-prioritized populations vaccinated before 31 July 2021 in three Swedish counties (total population 1,760,000). Subjects with a positive SARS-CoV-2 test recorded within 6 months before vaccination (n = 36,247; 6%) were matched to vaccinated-only controls. In the prioritized population exposed to the SARS-CoV-2 Alpha and Delta variants post-vaccination, the odds ratio (OR) for breakthrough infection was 2.2 (95% CI, 1.6−2.8; p < 0.001) in the vaccinated-only group compared with the hybrid immunity group, while in the later vaccinated non-prioritized population, the OR decreased from 4.3 (95% CI, 2.2−8.6; p < 0.001) during circulation of the Delta variant to 1.9 (95% CI, 1.7−2.1; p < 0.001) with the introduction of the Omicron variant (B.1.617.2). We conclude that hybrid immunity provides gains in protection, but that the benefits are smaller for risk groups and with circulation of the Omicron variant and its sublineages.

In this preclinical two-dose mucosal immunization study, using a combination of S1 spike and nucleocapsid proteins with cationic (N3)/or anionic (L3) lipids were investigated using an intranasal delivery route. The study showed that nasal administration of low amounts of antigens/adjuvants induced a primary and secondary immune response in systemic IgG, mIL-5, and IFN-gamma secreting T lymphocytes, as well as humoral IgA in nasal and intestinal mucosal compartments. It is believed that recipients will benefit from receiving a combination of viral antigens in promoting a border immune response against present and evolving contagious viruses. Lipid adjuvants demonstrated an enhanced response in the vaccine effect. This was seen in the significant immunogenicity effect when using the cationic lipid N3. Unlike L3, which showed a recognizable effect when administrated at a slightly higher concentration. Moreover, the findings of the study proved the efficiency of an intranasally mucosal immunization strategy, which can be less painful and more effective in enhancing the respiratory tract immunity against respiratory infectious diseases.

The use of nanoparticles for developing vaccines has become a routine process for researchers and pharmaceutical companies. Gold nanoparticles (GNPs) are chemical inert, have low toxicity, and are easy to modify and functionalize, making them an attractive choice for nanovaccine development. GNPs are modified for diagnostics and detection of many pathogens. The biocompatibility and biodistribution properties of GNPs render them ideal for use in clinical settings. They have excellent immune modulatory and adjuvant properties. They have been used as the antigen carrier for the delivery system to a targeted site. Tagging them with antibodies can direct the drug or antigen-carrying GNPs to specific tissues or cells. The physicochemical properties of the GNP, together with its dynamic immune response based on its size, shape, surface charge, and optical properties, make it a suitable candidate for vaccine development. The clear outcome of modulating dendritic cells, T and B lymphocytes, which trigger cytokine release in the host, indicates GNPs' efficiency in combating pathogens. The high titer of IgG and IgA antibody subtypes and their enhanced capacity to neutralize pathogens are reported in multiple studies on GNP-based vaccine development. The major focus of this review is to illustrate the role of GNPs in developing nanovaccines against multiple infectious agents, ranging from viruses to bacteria and parasites. Although the use of GNPs has its shortcomings and a low but detectable level of toxicity, their benefits warrant investing more thought and energy into the development of novel vaccine strategies.

Objectives: Impact of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic on individuals with arthritis has been highlighted whereas data on other rheumatic diseases, e.g., systemic lupus erythematosus (SLE), are scarce. Similarly to SLE, severe SARS-CoV-2 infection includes risks for thromboembolism, an unbalanced type I interferon response, and complement activation. Herein, SARS-CoV-2 antibodies in longitudinal samples collected prior to vaccination were analyzed and compared with SLE progression and antinuclear antibody (ANA) levels., Methods: One hundred patients (83 women) with established SLE and a regular visit to the rheumatologist (March 2020 to January 2021) were included. All subjects donated blood and had done likewise prior to the pandemic. SARS-CoV-2 antibody isotypes (IgG, IgA, IgM) to the cell receptor-binding S1-spike outer envelope protein were detected by ELISA, and their neutralizing capacity was investigated. IgG-ANA were measured by multiplex technology., Results: During the pandemic, 4% had PCR-confirmed infection but 36% showed SARS-CoV-2 antibodies of ≥1 isotype; IgA was the most common (30%), followed by IgM (9%) and IgG (8%). The antibodies had low neutralizing capacity and were detected also in prepandemic samples. Plasma albumin ( p = 0.04) and anti-dsDNA ( p = 0.003) levels were lower in patients with SARS-CoV-2 antibodies. Blood group, BMI, smoking habits, complement proteins, daily glucocorticoid dose, use of hydroxychloroquine, or self-reported coronavirus disease 2019 (COVID-19) symptoms (except fever, >38.5°C) did not associate with SARS-CoV-2 antibodies., Conclusion: Our data from early 2021 indicate that a large proportion of Swedish SLE patients had serological signs of exposure to SARS-CoV-2 but apparently with a minor impact on the SLE course. Use of steroids and hydroxychloroquine showed no distinct effects, and self-reported COVID-19-related symptoms correlated poorly with all antibody isotypes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sjöwall, Azharuddin, Frodlund, Zhang, Sandner, Dahle, Hinkula and Sjöwall.)

Nanomedicine is seen as a potential central player in the delivery of personalized medicine. Biocompatibility issues of nanoparticles have largely been resolved over the past decade. Despite their tremendous progress, less than 1% of applied nanosystems can hit their intended target location, such as a solid tumor, and this remains an obstacle to their full ability and potential with a high translational value. Therefore, achieving immune-tolerable, blood-compatible, and biofriendly nanoparticles remains an unmet need. The translational success of nanoformulations from bench to bedside involves a thorough assessment of their design, compatibility beyond cytotoxicity such as immune toxicity, blood compatibility, and immune-mediated destruction/rejection/clearance profile. Here, we report a one-pot process-engineered synthesis of ultrasmall gold nanoparticles (uGNPs) suitable for better body and renal clearance delivery of their payloads. We have obtained uGNP sizes of as low as 3 nm and have engineered the synthesis to allow them to be accurately sized (almost nanometer by nanometer). The synthesized uGNPs are biocompatible and can easily be functionalized to carry drugs, peptides, antibodies, and other therapeutic molecules. We have performed in vitro cell viability assays, immunotoxicity assays, inflammatory cytokine analysis, a complement activation study, and blood coagulation studies with the uGNPs to confirm their safety. These can help to set up a long-term safety-benefit framework of experimentation to reveal whether any designed nanoparticles are immune-tolerable and can be used as payload carriers for next-generation vaccines, chemotherapeutic drugs, and theranostic agents with better body clearance ability and deep tissue penetration.

One of the hallmarks of cancers is their ability to develop resistance against therapeutic agents. Therefore, developing effective in vitro strategies to identify drug resistance remains of paramount importance for successful treatment. One of the ways cancer cells achieve drug resistance is through the expression of efflux pumps that actively pump drugs out of the cells. To date, several studies have investigated the potential of using 3-dimensional (3D) multicellular tumor spheroids (MCSs) to assess drug resistance; however, a unified system that uses MCSs to differentiate between multi drug resistance (MDR) and non-MDR cells does not yet exist. In the present report we describe MCSs obtained from post-diagnosed, pre-treated patient-derived (PTPD) cell lines from head and neck squamous cancer cells (HNSCC) that often develop resistance to therapy. We employed an integrated approach combining response to clinical drugs and screening cytotoxicity, monitoring real-time drug uptake, and assessing transporter activity using flow cytometry in the presence and absence of their respective specific inhibitors. The report shows a comparative response to MDR, drug efflux capability and reactive oxygen species (ROS) activity to assess the resistance profile of PTPD MCSs and two-dimensional (2D) monolayer cultures of the same set of cell lines. We show that MCSs provide a robust and reliable in vitro model to evaluate clinical relevance. Our proposed strategy can also be clinically applicable for profiling drug resistance in cancers with unknown resistance profiles, which consequently can indicate benefit from downstream therapy.

Background : Vaccination is commonly used to prevent and control influenza infection in humans. However, improvements in the ease of delivery and strength of immunogenicity could markedly improve herd immunity. The aim of this pre-clinical study is to test the potential improvements to existing intranasal delivery of formalin-inactivated whole Influenza A vaccines (WIV) by formulation with a cationic lipid-based adjuvant (N3). Additionally, we combined WIV and N3 with a DNA-encoded TLR5 agonist secreted flagellin (pFliC(-gly)) as an adjuvant, as this adjuvant has previously been shown to improve the effectiveness of plasmid-encoded DNA antigens. Methods : Outbred and inbred mouse strains were intranasally immunized with unadjuvanted WIV A/H1N1/SI 2006 or WIV that was formulated with N3 alone. Additional groups were immunized with WIV and N3 adjuvant combined with pFliC(-gly). Homo and heterotypic humoral anti-WIV immune responses were assayed from serum and lung by ELISA and hemagglutination inhibition assay. Homo and heterotypic cellular immune responses to WIV and Influenza A NP were also determined. Results : WIV combined with N3 lipid adjuvant the pFliC(-gly) significantly increased homotypic influenza specific serum antibody responses (>200-fold), increased the IgG2 responses, indicating a mixed Th1/Th2-type immunity, and increased the HAI-titer (>100-fold). Enhanced cell-mediated IFNγ secreting influenza directed CD4 + and CD8 + T cell responses (>40-fold) to homotypic and heterosubtypic influenza A virus and peptides. Long-term and protective immunity was obtained. Conclusions : These results indicate that inactivated influenza virus that was formulated with N3 cationic adjuvant significantly enhanced broad systemic and mucosal influenza specific immune responses. These responses were broadened and further increased by incorporating DNA plasmids encoding FliC from S. typhimurum as an adjuvant providing long lasting protection against heterologous Influenza A/H1N1/CA09pdm virus challenge.

Because rotavirus (RV) and norovirus (NoV) are transmitted through the fecal-oral route, tonsils due to their location within the oropharynx may sample or become infected with these viruses. We investigated if RV and NoV RNA/antigen, or virus-specific memory/plasma B cells can be detected in the tonsils. While neither RV/NoV antigen, nor genomic RNA was detected, 90% (27/30) of tonsils tested had RV- and NoV-specific IgG memory B cells. However, the mechanism explaining how these cells get there (whether because of local induction or homing after induction at other sites) and the role these cells might play during active infection is not yet clear., (© 2018 Wiley Periodicals, Inc.)

The importance of natural IgM antibodies in protection against infections is still emerging and these antibodies have a potential role in the maintenance of homeostasis through clearance of apoptotic bodies, complement-dependent mechanisms, inflammation and exclusion of misfolded proteins. Natural IgM act as a first line of defence against unknown hazardous factors and are present in most vertebrates. We investigated the functional capacity of anti-HIV-1 IgM monoclonal antibodies, from a combinatorial Fab library derived from healthy individuals, and evaluated their protective role in inhibiting HIV-1 in vitro when passing across the human mucosal epithelial barrier. Primary HIV-1 isolates were efficiently transmitted over the tight polarized epithelial cells when added to their apical surface. Efficient inhibition of HIV-1 transmission was achieved when anti-HIV-1 IgM monoclonal antibodies were added to the basolateral side of the cells. Two of these human IgM MoAbs had the ability to neutralize HIV and reduced infection of dendritic cells in primary cervico-vaginal tissue biopsies in vitro. This indicates a potential role of natural IgM antibodies in the reduction of HIV-1 transmission in mucosal tissues and improve our understanding of how natural IgM antibodies against a neutralizing epitope could interfere with viral transmission.

Dendritic cells (DCs), natural killer (NK) cells, and T cells play critical roles during primary HIV-1 exposure at the mucosa, where the viral particles become coated with complement fragments and mucosa-associated antibodies. The microenvironment together with subsequent interactions between these cells and HIV at the mucosal site of infection will determine the quality of immune response that ensues adaptive activation. Here, we investigated how complement and immunoglobulin opsonization influences the responses triggered in DCs and NK cells, how this affects their cross talk, and what T cell phenotypes are induced to expand following the interaction. Our results showed that DCs exposed to complement-opsonized HIV (C-HIV) were less mature and had a poor ability to trigger IFN-driven NK cell activation. In addition, when the DCs were exposed to C-HIV, the cytotolytic potentials of both NK cells and CD8 T cells were markedly suppressed. The expression of PD-1 as well as co-expression of negative immune checkpoints TIM-3 and LAG-3 on PD-1 positive cells were increased on both CD4 as well as CD8 T cells upon interaction with and priming by NK-DC cross talk cultures exposed to C-HIV. In addition, stimulation by NK-DC cross talk cultures exposed to C-HIV led to the upregulation of CD38, CXCR3, and CCR4 on T cells. Together, the immune modulation induced during the presence of complement on viral surfaces is likely to favor HIV establishment, dissemination, and viral pathogenesis.

The zinc finger CCCH-type containing 11A ( ZC3H11A ) gene encodes a well-conserved zinc finger protein that may function in mRNA export as it has been shown to associate with the transcription export (TREX) complex in proteomic screens. Here, we report that ZC3H11A is a stress-induced nuclear protein with RNA-binding capacity that localizes to nuclear splicing speckles. During an adenovirus infection, the ZC3H11A protein and splicing factor SRSF2 relocalize to nuclear regions where viral DNA replication and transcription take place. Knockout (KO) of ZC3H11A in HeLa cells demonstrated that several nuclear-replicating viruses are dependent on ZC3H11A for efficient growth (HIV, influenza virus, herpes simplex virus, and adenovirus), whereas cytoplasmic replicating viruses are not (vaccinia virus and Semliki Forest virus). High-throughput sequencing of ZC3H11A-cross-linked RNA showed that ZC3H11A binds to short purine-rich ribonucleotide stretches in cellular and adenoviral transcripts. We show that the RNA-binding property of ZC3H11A is crucial for its function and localization. In ZC3H11A KO cells, the adenovirus fiber mRNA accumulates in the cell nucleus. Our results suggest that ZC3H11A is important for maintaining nuclear export of mRNAs during stress and that several nuclear-replicating viruses take advantage of this mechanism to facilitate their replication., Competing Interests: Conflict of interest statement: S.Y., W.K., G.A., and L.A. are coauthors on a patent application filed based on some of the results in this study., (Copyright © 2018 the Author(s). Published by PNAS.)

Crimean-Congo hemorrhagic fever virus (CCHFV) is a bunyavirus causing severe hemorrhagic fever disease in humans, with high mortality rates. The requirement of a high-containment laboratory and the lack of an animal model hampered the study of the immune response and protection of vaccine candidates. Using the recently developed interferon alpha receptor knockout (IFNAR -/- ) mouse model, which replicates human disease, we investigated the immunogenicity and protection of two novel CCHFV vaccine candidates: a DNA vaccine encoding a ubiquitin-linked version of CCHFV Gc, Gn, and N and one using transcriptionally competent virus-like particles (tc-VLPs). In contrast to most studies that focus on neutralizing antibodies, we measured both humoral and cellular immune responses. We demonstrated a clear and 100% efficient preventive immunity against lethal CCHFV challenge with the DNA vaccine. Interestingly, there was no correlation with the neutralizing antibody titers alone, which were higher in the tc-VLP-vaccinated mice. However, the animals with a lower neutralizing titer, but a dominant cell-mediated Th1 response and a balanced Th2 response, resisted the CCHFV challenge. Moreover, we found that in challenged mice with a Th1 response (immunized by DNA/DNA and boosted by tc-VLPs), the immune response changed to Th2 at day 9 postchallenge. In addition, we were able to identify new linear B-cell epitope regions that are highly conserved between CCHFV strains. Altogether, our results suggest that a predominantly Th1-type immune response provides the most efficient protective immunity against CCHFV challenge. However, we cannot exclude the importance of the neutralizing antibodies as the surviving immunized mice exhibited substantial amounts of them. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is responsible for hemorrhagic diseases in humans, with a high mortality rate. There is no FDA-approved vaccine, and there are still gaps in our knowledge of the immune responses to infection. The recently developed mouse models mimic human CCHF disease and are useful to study the immunogenicity and the protection by vaccine candidates. Our study shows that mice vaccinated with a specific DNA vaccine were fully protected. Importantly, we show that neutralizing antibodies are not sufficient for protection against CCHFV challenge but that an extra Th1-specific cellular response is required. Moreover, we describe the identification of five conserved B-cell epitopes, of which only one was previously known, that could be of great importance for the development of diagnostics tools and the improvement of vaccine candidates., (Copyright © 2017 Hinkula et al.)

Despite availability of annual influenza vaccines, influenza causes significant morbidity and mortality in the elderly. This is at least in part a result of immunosenescence; the age-dependent decrease in immunological competence that results in greater susceptibility to infections and reduced responses to vaccination. To improve protective immune responses in this age group, new vaccines strategies, such as the use of adjuvants, are needed. Here, we evaluated the mucosal vaccine adjuvant Endocine™, formulated with split influenza antigen and administered intranasally in aged (20-month old) mice. Humoral immune responses were assessed and compared to unadjuvanted intranasal and subcutaneous vaccines. We show that formulation with Endocine™ significantly enhances hemagglutination inhibition (HI) titers, as well as serum IgG and mucosal IgA antibody titers, compared to both types of unadjuvanted vaccines. Thus, our results indicate that intranasal vaccination with Endocine™ is a possible approach for the development of mucosal influenza vaccines for the elderly.

Reverse transcriptase (RT) is a key enzyme in viral replication and susceptibility to ART and a crucial target of immunotherapy against drug-resistant HIV-1. RT induces oxidative stress which undermines the attempts to make it immunogenic. We hypothesized that artificial secretion may reduce the stress and make RT more immunogenic. Inactivated multidrug-resistant RT (RT1.14opt-in) was N-terminally fused to the signal providing secretion of NS1 protein of TBEV (Ld) generating optimized inactivated Ld-carrying enzyme RT1.14oil. Promotion of secretion prohibited proteasomal degradation increasing the half-life and content of RT1.14oil in cells and cell culture medium, drastically reduced the residual polymerase activity, and downmodulated oxidative stress. BALB/c mice were DNA-immunized with RT1.14opt-in or parental RT1.14oil by intradermal injections with electroporation. Fluorospot and ELISA tests revealed that RT1.14opt-in and RT1.14oil induced IFN- γ /IL-2, RT1.14opt-in induced granzyme B, and RT1.14oil induced perforin production. Perforin secretion correlated with coproduction of IFN- γ and IL-2 ( R = 0,97). Both DNA immunogens induced strong anti-RT antibody response. Ld peptide was not immunogenic. Thus, Ld-driven secretion inferred little change to RT performance in DNA immunization. Positive outcome was the abrogation of polymerase activity increasing safety of RT-based DNA vaccines. Identification of the molecular determinants of low cellular immunogenicity of RT requires further studies.

Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Immature dendritic cells (iDCs) in genital and rectal mucosa may be one of the first cells to come into contact with HIV-1 during sexual transmission of virus. HIV-1 activates the host complement system, which results in opsonization of virus by inactivated complement fragments, for example, iC3b. We investigated antiviral and inflammatory responses induced in human iDCs after exposure to free HIV-1 (F-HIV), complement-opsonized HIV-1 (C-HIV), and complement and Ab-opsonized HIV-1 (CI-HIV). F-HIV gave rise to a significantly higher expression of antiviral factors such as IFN-β, myxovirus resistance protein A, and IFN-stimulated genes, compared with C-HIV and CI-HIV. Additionally, F-HIV induced inflammatory factors such as IL-1β, IL-6, and TNF-α, whereas these responses were weakened or absent after C-HIV or CI-HIV exposure. The responses induced by F-HIV were TLR8-dependent with subsequent activation of IFN regulatory factor 1, p38, ERK, PI3K, and NF-κB pathways, whereas these responses were not induced by C-HIV, which instead induced activation of IFN regulatory factor 3 and Lyn. This modulation of TLR8 signaling was mediated by complement receptor 3 and led to enhanced infection. The impact that viral hijacking of the complement system has on iDC function could be an important immune evasion mechanism used by HIV-1 to establish infection in the host., (Copyright © 2014 by The American Association of Immunologists, Inc.)

DNA vaccines based on subunits from pathogens have several advantages over other vaccine strategies. DNA vaccines can easily be modified, they show good safety profiles, are stable and inexpensive to produce, and the immune response can be focused to the antigen of interest. However, the immunogenicity of DNA vaccines which is generally quite low needs to be improved. Electroporation and co-delivery of genetically encoded immune adjuvants are two strategies aiming at increasing the efficacy of DNA vaccines. Here, we have examined whether targeting to antigen-presenting cells (APC) could increase the immune response to surface envelope glycoprotein (Env) gp120 from Human Immunodeficiency Virus type 1 (HIV-1). To target APC, we utilized a homodimeric vaccine format denoted vaccibody, which enables covalent fusion of gp120 to molecules that can target APC. Two molecules were tested for their efficiency as targeting units: the antibody-derived single chain Fragment variable (scFv) specific for the major histocompatibility complex (MHC) class II I-E molecules, and the CC chemokine ligand 3 (CCL3). The vaccines were delivered as DNA into muscle of mice with or without electroporation. Targeting of gp120 to MHC class II molecules induced antibodies that neutralized HIV-1 and that persisted for more than a year after one single immunization with electroporation. Targeting by CCL3 significantly increased the number of HIV-1 gp120-reactive CD8+ T cells compared to non-targeted vaccines and gp120 delivered alone in the absence of electroporation. The data suggest that chemokines are promising molecular adjuvants because small amounts can attract immune cells and promote immune responses without advanced equipment such as electroporation.

Influenza is a contagious respiratory disease caused by an influenza virus. Due to continuous antigenic drift of seasonal influenza viruses, influenza vaccines need to be adjusted before every influenza season. This allows annual vaccination with multivalent seasonal influenza vaccines, recommended especially for high-risk groups. There is a need for a seasonal influenza vaccine that induces broader and longer lasting protection upon easy administration. Endocine is a lipid-based mucosal adjuvant composed of endogenous lipids found ubiquitously in the human body. Intranasal administration of influenza antigens mixed with this adjuvant has been shown to induce local and systemic immunity as well as protective efficacy against homologous influenza virus challenge in mice. Here we used ferrets, an established animal model for human influenza virus infections, to further investigate the potential of Endocine as an adjuvant. Intranasal administration of inactivated pandemic H1N1/California/2009 split antigen or whole virus antigen mixed with Endocine induced high levels of serum hemagglutination inhibition (HI) and virus neutralization (VN) antibody titers that were also cross reactive against distant swine viruses of the same subtype. HI and VN antibody titers were already demonstrated after a single nasal immunization. Upon intratracheal challenge with a homologous challenge virus (influenza virus H1N1/The Netherlands/602/2009) immunized ferrets were fully protected from virus replication in the lungs and largely protected against body weight loss, virus replication in the upper respiratory tract and pathological changes in the respiratory tract. Endocine formulated vaccines containing split antigen induced higher HI and VN antibody responses and better protection from body weight loss and virus shedding in the upper respiratory tract than the Endocine formulated vaccine containing whole virus antigen., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: There is abundant global interest in using syndromic data from population-wide health information systems--referred to as eHealth resources--to improve infectious disease surveillance. Recently, the necessity for these systems to achieve two potentially conflicting requirements has been emphasized. First, they must be evidence-based; second, they must be adjusted for the diversity of populations, lifestyles, and environments., Objective: The primary objective was to examine correlations between data from Google Flu Trends (GFT), computer-supported telenursing centers, health service websites, and influenza case rates during seasonal and pandemic influenza outbreaks. The secondary objective was to investigate associations between eHealth data, media coverage, and the interaction between circulating influenza strain(s) and the age-related population immunity., Methods: An open cohort design was used for a five-year study in a Swedish county (population 427,000). Syndromic eHealth data were collected from GFT, telenursing call centers, and local health service website visits at page level. Data on mass media coverage of influenza was collected from the major regional newspaper. The performance of eHealth data in surveillance was measured by correlation effect size and time lag to clinically diagnosed influenza cases., Results: Local media coverage data and influenza case rates showed correlations with large effect sizes only for the influenza A (A) pH1N1 outbreak in 2009 (r=.74, 95% CI .42-.90; P<.001) and the severe seasonal A H3N2 outbreak in 2011-2012 (r=.79, 95% CI .42-.93; P=.001), with media coverage preceding case rates with one week. Correlations between GFT and influenza case data showed large effect sizes for all outbreaks, the largest being the seasonal A H3N2 outbreak in 2008-2009 (r=.96, 95% CI .88-.99; P<.001). The preceding time lag decreased from two weeks during the first outbreaks to one week from the 2009 A pH1N1 pandemic. Telenursing data and influenza case data showed correlations with large effect sizes for all outbreaks after the seasonal B and A H1 outbreak in 2007-2008, with a time lag decreasing from two weeks for the seasonal A H3N2 outbreak in 2008-2009 (r=.95, 95% CI .82-.98; P<.001) to none for the A p H1N1 outbreak in 2009 (r=.84, 95% CI .62-.94; P<.001). Large effect sizes were also observed between website visits and influenza case data., Conclusions: Correlations between the eHealth data and influenza case rates in a Swedish county showed large effect sizes throughout a five-year period, while the time lag between signals in eHealth data and influenza rates changed. Further research is needed on analytic methods for adjusting eHealth surveillance systems to shifts in media coverage and to variations in age-group related immunity between virus strains. The results can be used to inform the development of alert-generating eHealth surveillance systems that can be subject for prospective evaluations in routine public health practice.

Failure to incorporate the beliefs and attitudes of the public into theoretical models of preparedness has been identified as a weakness in strategies to mitigate infectious disease outbreaks. We administered a cross-sectional telephone survey to a representative sample (n = 443) of the Swedish adult population to examine whether self-reported intentions to improve personal hygiene and increase social distancing during influenza outbreaks could be explained by trust in official information, self-reported health (SF-8), sociodemographic factors, and determinants postulated in protection motivation theory, namely threat appraisal and coping appraisal. The interviewees were asked to make their appraisals for two scenarios: a) an influenza with low case fatality and mild lifestyle impact; b) severe influenza with high case fatality and serious disturbances of societal functions. Every second respondent (50.0%) reported high trust in official information about influenza. The proportion that reported intentions to take deliberate actions to improve personal hygiene during outbreaks ranged between 45-85%, while less than 25% said that they intended to increase social distancing. Multiple logistic regression models with coping appraisal as the explanatory factor most frequently contributing to the explanation of the variance in intentions showed strong discriminatory performance for staying home while not ill (mild outbreaks: Area under the curve [AUC] 0.85 (95% confidence interval 0.82;0.89), severe outbreaks AUC 0.82 (95% CI 0.77;0.85)) and acceptable performance with regard to avoiding public transportation (AUC 0.78 (0.74;0.82), AUC 0.77 (0.72;0.82)), using handwash products (AUC 0.70 (0.65;0.75), AUC 0.76 (0.71;0.80)), and frequently washing hands (AUC 0.71 (0.66;0.76), AUC 0.75 (0.71;0.80)). We conclude that coping appraisal was the explanatory factor most frequently included in statistical models explaining self-reported intentions to carry out non-pharmaceutical health actions in the Swedish outlined context, and that variations in threat appraisal played a smaller role in these models despite scientific uncertainties surrounding a recent mass vaccination campaign.

Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge. We have previously shown that ballistic dermal plasmid DNA-encoded flagellin (FliC) promotes humoral as well as cellular immunity to co-delivered antigens. Here, we observe that a plasmid encoding secreted FliC (pFliC(-gly)) produces flagellin capable of activating two innate immune receptors known to detect flagellin; Toll-like Receptor 5 (TLR5) and Nod-like Receptor family CARD domain-containing protein 4 (NRLC4). To test the ability of pFliC(-gly) to act as an adjuvant we immunized mice with plasmid encoding secreted FliC (pFliC(-gly)) and plasmid encoding a model antigen (ovalbumin) by three different immunization routes representative of dermal, systemic, and mucosal tissues. By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added. Additionally, we were able to induce mucosal antibody responses and Class II-dependent cellular immune responses after mucosal vaccination with pFliC(-gly). Humoral immune responses elicited by heterologus prime-boost immunization with a plasmid encoding HIV-1 from gp160 followed by protein boosting could be enhanced by use of pFliC(-gly). We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity. These observations indicate that plasmid-encoded secreted flagellin can activate multiple innate immune responses and function as an adjuvant to non-living/replicating DNA immunizations. Moreover, the capacity to elicit mucosal immune responses, in addition to dermal and systemic properties, demonstrates the potential of flagellin to be used with vaccines designed to be delivered by various routes.

The initial interaction between HIV-1 and the host occurs at the mucosa during sexual intercourse. In cervical mucosa, HIV-1 exists both as free and opsonized virions and this might influence initial infection. We used cervical explants to study HIV-1 transmission, the effects of opsonization on infectivity, and how infection can be prevented. Complement opsonization enhanced HIV-1 infection of dendritic cells (DCs) compared with that by free HIV-1, but this increased infection was not observed with CD4(+) T cells. Blockage of the α4-, β7-, and β1-integrins significantly inhibited HIV-1 infection of both DCs and CD4(+) T cells. We found a greater impairment of HIV-1 infection in DCs for complement-opsonized virions compared with that of free virions when αM/β2- and α4-integrins were blocked. Blocking the C-type lectin receptor macrophage mannose receptor (MMR) inhibited infection of emigrating DCs but had no effect on CD4(+) T-cell infection. We show that blocking of integrins decreases the HIV-1 infection of both mucosal DCs and CD4(+) T cells emigrating from the cervical tissues. These findings may provide the basis of novel microbicidal strategies that may help limit or prevent initial infection of the cervical mucosa, thereby reducing or averting systemic HIV-1 infection., (© 2013 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA Weinheim.)

Annual outbreaks of seasonal influenza are controlled or prevented through vaccination in many countries. The seasonal vaccines used are either inactivated, currently administered parenterally, or live-attenuated given intranasally. In this study three mucosal adjuvants were examined for the influence on the humoral (mucosal and systemic) and cellular influenza A-specific immune responses induced by a nasally administered vaccine. We investigated in detail how the anionic Endocine™ and the cationic adjuvants N3OA and N3OASq mixed with a split inactivated influenza vaccine induced influenza A-specific immune responses as compared to the vaccine alone after intranasal immunization. The study showed that nasal administration of a split virus vaccine together with Endocine™ or N3OA induced significantly higher humoral and cell-mediated immune responses than the non-adjuvanted vaccine. N3OASq only significantly increased the cell-mediated immune response. Furthermore, nasal administration of the influenza vaccine in combination with any of the adjuvants; Endocine™, N3OA or N3OASq, significantly enhanced the mucosal immunity against influenza HA protein. Thus the addition of these mucosal adjuvants leads to enhanced immunity in the most relevant tissues, the upper respiratory tract and the systemic circulation. Nasal influenza vaccination with an inactivated split vaccine can therefore provide an important mucosal immune response, which is often low or absent after traditional parenteral vaccination.

Induction of optimal HIV-1-specific T-cell responses, which can contribute to controlling viral infection in vivo, depends on antigen processing and presentation processes occurring in DCs. Opsonization can influence the routing of antigen processing and pathways used for presentation. We studied antigen proteolysis and the role of endocytic receptors in MHC class I (MHCI) and II (MHCII) presentation of antigens derived from HIV-1 in human monocyte-derived immature DCs (IDCs) and mature DCs, comparing free and complement opsonized HIV-1 particles. Opsonization of virions promoted MHCI presentation by DCs, indicating that complement opsonization routes more virions toward the MHCI presentation pathway. Blockade of macrophage mannose receptor (MMR) and β7-integrin enhanced MHCI and MHCII presentation by IDCs and mature DCs, whereas the block of complement receptor 3 decreased MHCI and MHCII presentation. In addition, we found that IDC and MDC proteolytic activities were modulated by HIV-1 exposure; complement-opsonized HIV-1 induced an increased proteasome activity in IDCs. Taken together, these findings indicate that endocytic receptors such as MMR, complement receptor 3, and β7-integrin can promote or disfavor antigen presentation probably by routing HIV-1 into different endosomal compartments with distinct efficiencies for degradation of viral antigens and MHCI and MHCII presentation, and that HIV-1 affects the antigen-processing machinery., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Preclinical vaccine efficacy studies are generally limited to certain read out parameters such as assessment of virus titers in swabs and organs, clinical signs, serum antibody titers, and pathological changes. These parameters are not always routinely applied and not always scheduled in a logical standardized way. We used computed tomography (CT) imaging as additional and novel read out parameter in a vaccine efficacy study by quantifying alterations in aerated lung volumes in ferrets challenged with the 2009 pandemic A/H1N1 influenza virus. Vaccination protected from marked variations in aerated lung volumes compared to naive controls. The vaccinated group showed a daily gradual mean reduction with a maximum of 7.8%, whereas the controls showed a maximum of 14.3% reduction. The pulmonary opacities evident on CT images were most pronounced in the placebo-treated controls, and corresponded to significantly increased relative lung weights at necropsy. This study shows that consecutive in vivo CT imaging allows for a day to day read out of vaccine efficacy by quantification of altered aerated lung volumes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human immunodeficiency virus type 1 (HIV-1) infection enhances the expression of inhibitory molecules on T cells, leading to T-cell impairment. The signaling pathways underlying the regulation of inhibitory molecules and subsequent onset of T-cell impairment remain elusive. We showed that both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells (DCs) upregulated cytotoxic T-lymphocyte antigen (CTLA-4), tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), lymphocyte-activation gene-3 (LAG3), T-cell immunoglobulin mucin-3 (TIM-3), CD160 and certain suppression-associated transcription factors, such as B-lymphocyte induced maturation protein-1 (BLIMP-1), deltex homolog 1 protein (DTX1) and forkhead box P3 (FOXP3), leading to T-cell suppression. This induction was regulated by p38 mitogen-activated protein kinase/signal transducer and activator of transcription-3 (P38MAPK/STAT3) pathways, because their blockade significantly abrogated expression of all the inhibitory molecules studied and a subsequent recovery in T-cell proliferation. Neither interleukin-6 (IL-6) nor IL-10 nor growth factors known to activate STAT3 signaling events were responsible for STAT3 activation. Involvement of the P38MAPK/STAT3 pathways was evident because these proteins had a higher level of phosphorylation in the HIV-1-primed cells. Furthermore, blockade of viral CD4 binding and fusion significantly reduced the negative effects DCs imposed on primed T cells. In conclusion, HIV-1 interaction with DCs modulated their functionality, causing them to trigger the activation of the P38MAPK/STAT3 pathway in T cells, which was responsible for the upregulation of inhibitory molecules.

We increase our understanding of augmenting a cellular immune response, by using an HIV-1 protease-derived epitope (PR75-84), and variants thereof, coupled to the C-terminal, of the B subunit of cholera toxin (CTB). Fusion proteins were used for immunizations of HLA-A0201 transgenic C57BL/6 mice. We observed different capacities to elicit a cellular immune response by peptides with additions of five to ten amino acids to the PR epitope. There was a positive correlation between the magnitude of the elicited cellular immune response and the capacity of the fusion protein to bind GM-1. This binding capacity is affected by its ability to form natural pentamers of CTB. Our results suggest that functional CTB pentamers containing a foreign amino acid-modified epitope is a novel way to overcome the limited cellular immunogenicity of minimal peptide antigens. This way of using a functional assay as readout for improved cellular immunogenicity might become highly valuable for difficult immunogens such as short peptides (epitopes).

Female rhesus macaques were immunized with HIV virus-like particles (HIV-VLPs) or HIV DNA administered as sequential combinations of mucosal (intranasal) and systemic (intramuscular) routes, according to homologous or heterologous prime-boost schedules. The results show that in rhesus macaques only the sequential intranasal and intramuscular administration of HIV-VLPs, and not the intranasal alone, is able to elicit humoral immune response at the systemic as well as the vaginal level.

We constructed novel HIV-1 fusion inhibitors that may overcome the current limitations of enfuvirtide, the first such therapeutic in this class. The three prototypes generated by the Dock-and-Lock (DNL) technology to comprise four copies of enfuvirtide tethered site-specifically to the Fc end of different humanized monoclonal antibodies potently neutralize primary isolates (both R5-tropic and X4-tropic), as well as T-cell-adapted strains of HIV-1 in vitro. All three prototypes show EC(50) values in the subnanomolar range, which are 10- to 100-fold lower than enfuvirtide and attainable whether or not the constitutive antibody targets HIV-1. The potential of such conjugates to purge latently infected cells was also demonstrated in a cell-to-cell viral inhibition assay by measuring their efficacy to inhibit the spread of HIV-1(LAI) from infected human peripheral blood mononuclear cells to Jurkat T cells over a period of 30 days following viral activation with 100 nM SAHA (suberoylanilide hydroxamic acid). The IgG-like half-life was not significantly different from that of the parental antibody, as shown by the mean serum concentration of one prototype in mice at 72 h. These encouraging results provide a rationale to develop further novel anti-HIV agents by coupling additional antibodies of interest with alternative HIV-inhibitors via recombinantly-produced, self-assembling, modules.

Tuberculosis (TB) has emerged as the most prominent bacterial disease found in human immunodeficiency virus (HIV)-positive individuals worldwide. Due to high prevalence of asymptomatic Mycobacterium tuberculosis (Mtb) infections, the future HIV vaccine in areas highly endemic for TB will often be administrated to individuals with an ongoing Mtb infection. The impact of concurrent Mtb infection on the immunogenicity of a HIV vaccine candidate, MultiHIV DNA/protein, was investigated in mice. We found that, depending on the vaccination route, mice infected with Mtb before the administration of the HIV vaccine showed impairment in both the magnitude and the quality of antibody and T cell responses to the vaccine components p24Gag and gp160Env. Mice infected with Mtb prior to intranasal HIV vaccination exhibited reduced p24Gag-specific serum IgG and IgA, and suppressed gp160Env-specific serum IgG as compared to respective titers in uninfected HIV-vaccinated controls. Importantly, in Mtb-infected mice that were HIV-vaccinated by the intramuscular route the virus neutralizing activity in serum was significantly decreased, relative to uninfected counterparts. In addition mice concurrently infected with Mtb had fewer p24Gag-specific IFN-γ-expressing T cells and multifunctional T cells in their spleens. These results suggest that Mtb infection might interfere with the outcome of prospective HIV vaccination in humans.

Natural killer (NK) cells have been suggested to play a protective role in HIV disease progression. One potent effector mechanism of NK cells is antibody-dependent cellular cytotoxicity (ADCC) mediated by antiviral antibodies binding to the FcγRIIIa receptor (CD16) on NK cells. We investigated NK cell-mediated ADCC function and the presence of ADCC antibodies in plasma from 20 HIV-1-infected patients and 10 healthy donors. The HIV-positive patients were divided into two groups: six who controlled viremia for at least 8 y without treatment (controllers), and 14 who were persistently viremic and not currently on treatment. Plasma from both patient groups induced NK cell IFN-γ expression and degranulation in response to HIV-1 envelope (Env) gp140-protein-coated cells. Patient antibodies mediating ADCC were largely directed towards the Env V3 loop, as identified by a gp140 protein lacking the V3 loop. Interestingly, in two controllers ADCC-mediating antibodies were more broadly directed to other parts of Env. A high viral load in patients correlated with decreased ADCC-mediated cytolysis of gp140-protein-coated target cells. NK cells from both infected patients and healthy donors degranulated efficiently in the presence of antibody-coated HIV-1-infected Jurkat cells. In conclusion, the character of ADCC-mediating antibodies differed in some controllers compared to viremic patients. NK cell ADCC activity is not compromised in HIV-infected patients.

Natural killer (NK) cells are important effectors in resistance to viral infections. The role of NK cells in the acute response to human immunodeficiency virus 1 (HIV-1) infected cells was investigated in a mouse model based on a HIV-1/murine leukemia virus (MuLV) pseudovirus. Splenocytes infected with HIV-1/MuLV were injected intraperitoneally and local immunologic responses and persistence of infected cells were investigated. In vivo depletion with an anti-NK1.1 antibody showed that NK cells are important in resistance to virus infected cells. Moreover, NK cell frequency in the peritoneal cavity increased in response to infected cells and these NK cells had a more mature phenotype, as determined by CD27 and Mac-1 expression. Interestingly, after injection of HIV-1/MuLV infected cells, but not MuLV infected cells, peritoneal NK cells had an increased cytotoxic activity. In conclusion, NK cells play a role in the early control of HIV-1/MuLV infected cells in vivo., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Interaction with the complement system is an underappreciated aspect of HIV-1 infection; even in primary infection, complement fragments are found on virions with potential to affect the interplay between the virus and dendritic cells (DC). Since opsonization may affect the efficiency of uptake and the type of receptors utilized, we compared the interactions of DC with free HIV-1 (F-HIV) and complement opsonized HIV-1 (C-HIV). We demonstrate that C-HIV significantly enhanced the uptake by immature DC (IDC) and mature DC (MDC) and that the internalization rate was dependent on both opsonization of the virus and DC maturation state. Increased DC uptake of C-HIV was not due to opsonization related increased binding of virus to the surface of DC but rather increased internalization of C-HIV despite utilizing a similar repertoire of receptors as F-HIV. Both F-HIV and C-HIV interacted with C-type lectins, integrins, and CD4 and blocking these receptor families prevented HIV-1 from binding to DC at 4°C. Blocking integrins significantly reduced the binding and uptake of F-HIV and C-HIV implicating the involvement of several integrins such as β1-integrin, CR3, LFA-1, and α4β7. Distinctive for C-HIV was usage of β1-integrin and for F-HIV, usage of β7-integrin, whereas both F-HIV and C-HIV utilized both integrin chains of CR3. We have in this study identified the receptor types used by both F-HIV and C-HIV to bind to DC. Noteworthy, C-HIV was internalized more efficiently by DC than F-HIV, probably via receptor mediated endocytosis, which may entail different intracellular processing of the virus leading to both elevated infection and altered activation of HIV specific immune responses.

A key gap in the development and evaluation of HIV-1 vaccines is insufficient knowledge with regard to sampling techniques and assessment of mucosal immune responses required for early prevention and inhibition of viral dissemination. In an attempt to start bridging this gap, the EUROPRISE network of scientists working on HIV-1 vaccine and microbicide research organized a workshop with the aim to review the types of mucosal responses/biomarkers currently measured in mucosal immunology and to define how the mucosal responses/biomarkers are measured and/or the assays and sampling methods used. The Workshop addressed two critical questions: first whether, with current knowledge, it would be possible to define a consensus set of mucosal sampling methods to facilitate cross-species comparisons and ensure standardized implementation in clinical trials; second to determine the remaining challenges (technical and logistical) and their possible solutions for assessing mucosal responses to HIV-1 vaccines.

Delivery of DNA encoding foreign antigens into mammalian cells can induce adaptive immune responses. There are currently many DNA-based vaccines in clinical trials against infectious diseases and cancer but there is a lack of adjuvants for improvement of responses to DNA-based vaccines. Here, we show augmented systemic and mucosa-associated B cell responses after immunization with a cocktail of seven different plasmids (3 env, 2 gag, 1 rev, 1 RT) combined with mitogen activated apoptotic syngeneic lymphocytes in mice. In addition we show that apoptotic cells can function as adjuvant for induction of cellular immune responses in a magnitude comparable to the cytokine adjuvant GM-CSF in mice. These data suggest that activated apoptotic lymphocytes can act independent as adjuvants to improve antigen-specific DNA vaccines., (Copyright 2009 Elsevier Ltd. All rights reserved.)