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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).)

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.

Background: Genetic immunization is expected to induce the expression of antigens in a native form. The encoded peptide epitopes are presented on endogenous MHC molecules, mimicking antigen presentation during a viral infection. We have explored the potential of enfuvirtide (T20), a short HIV peptide with antiviral properties, to enhance immune response to HIV antigens. To generate an expression vector, the T20 sequence was cloned into a conventional plasmid, the novel minicircle construct, and a replicon plasmid. In addition, 3 conventional plasmids that express the envelope of HIV-1 subtypes A, B and C and contain T20 in their gp41 sequences were also tested., Results: All combinations induced HIV-specific antibodies and cellular responses. The addition of T20 as a peptide and as an expression cassette in the 3 DNA vectors enhanced antibody responses. The highest anti-HIV-1 Env titers were obtained by the replicon T20 construct. This demonstrates that besides its known antiviral activity, T20 promotes immune responses. We also confirm that the combination of slightly divergent antigens improves immune responses., Conclusions: The antiretroviral T20 HIV-1 sequence can be used as an immunogen to elicit binding and neutralizing antibodies against HIV-1. These, or similarly modified gp41 genes/peptides, can be used as priming or boosting components for induction of broadly neutralizing anti-HIV antibodies. Future comparative studies will reveal the optimal mode of T20 administration.

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.

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.)

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.)

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 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.

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.

This study was designed to determine the impact of maternal HIV-1 specific immunity on HIV-DNA immunization of 2-week-old pups during the breast-feeding period. Adult female mice received intranasal or intradermal HIV-DNA (gp160Env, p37Gag, Nef, Tat and Rev) prime and recombinant protein boost immunizations, which induced mucosal and systemic HIV-1 specific B and T cell responses. Intranasal administration of the immunogens induced higher serum IgG titers to HIV antigens than intradermal immunization. Furthermore, high HIV-1 specific fecal IgA titers were obtained in mice immunized by intranasal administration. The capacity to respond to the same immunogens (one single prime with DNA and one boost with recombinant protein) was then compared in pups born to mothers with HIV-1-specific immune responses and pups born to non-vaccinated mothers. Immune responses to the largest number of antigens were detected in pups born to mothers with the highest HIV-1-specific immune responses. These data show that HIV-1 DNA-plasmid immunization during breast-feeding and recombinant protein boosting shortly thereafter enhance the breadth of humoral HIV-1-specific immune responses.

Development of transgenic edible plants, to be used as production, storage and delivery systems for recombinant vaccine antigens, is a promising strategy to obtain cost effective vaccines against infectious diseases, not least for use in developing countries. Therefore, we used Agrobacterium tumefaciens-mediated gene transfer to introduce the p24 gag gene encoding the nucleocapsid protein from HIV-1 subtype C into the Arabidopsis thaliana plant genome. Eighteen plant lines were confirmed positive for the p24 gene by PCR; four of these lines showed an apparent homozygous phenotype when grown on selective medium and these lines also showed transcription of the p24 gene into its corresponding mRNA. The mRNA in all four cases generated the p24 protein in plants, as verified by Western blot analysis. The plants were shown to contain between 0.2 mug and 0.5 mug p24 protein per g of fresh tissue. Analysis of the localisation of the p24 protein showed that stem tissue contained the largest amount of protein, more than twice as much as leaf tissue, whereas no p24 protein was detected in roots. By using Southern blotting, we found that 4, 2-3, 2 and 1 T-DNA insertion events took place in the four lines 1, 2, 7, and 10, respectively. The genetic insertions of line 1 were stable from the T(2) to the T(5) generation and gave rise to the p24 protein in all cases, as verified by Western blotting. In mice fed with fresh transgenic A. thaliana (line 10), anti-gag IgG was obtained in serum after a booster injection with recombinant p37Gag. No immune response was observed after equal booster injection of untreated mice or mice fed with A. thaliana WT plants.

Recent results by us and others have shown that the accessory protein Vpu determines plasma membrane versus endosomal accumulation of the HIV-1 core protein Gag and progeny virions in the HeLa model of HIV-1 infection, since Vpu suppresses endocytosis of cell surface-associated Gag. In this report, we used pulse-chase studies and subcellular fractionations to investigate endocytosis of newly synthesized Gag in HeLa H1 cells. The uptake of Gag in Delta Vpu-virus background was not blocked by inhibitors of clathrin-mediated endocytosis and macropinocytosis. The cholesterol-sequestering drug filipin inhibited the uptake, but only if the drug was applied before extensive multimerization of Gag had taken place. Thus, the uptake mechanism most likely is only indirectly dependent on cholesterol. Our results also indicated that targeting phenotype of Gag was different in confluent versus subconfluent cell cultures, which could perhaps explain some of the controversies in intracellular targeting of Gag.

Pre-clinical HIV-1 vaccine protocols, using multiple vaccine modalities and a potent adjuvant were assessed for vaccine efficacy in an experimental HIV-1 challenge model. C57Bl/6 mice were immunized with DNA plasmids encoding HIV-1 gp140, Gag and Tat alone or in combination with the corresponding recombinant proteins formulated in the adjuvant MF59. HIV-1 DNA alone or a DNA prime protein boost schedule resulted in complete protection against challenge with HIV-1/MuLV-infected murine cells. Although HIV-1 protein immunization in combination with MF59 resulted in partial protection, the DNA priming seemed to be crucial for obtaining full protection against the challenge. It is likely that the partial protection seen after immunization with protein alone is, to a certain extent, due to effects of the adjuvant since some animals that received the adjuvant MF59 alone were protected from the challenge. For the most part, antigen-specific cell-mediated immune responses as detected in the spleen (in contrast to responses detected in peripheral blood) of immunized animals appeared to be associated with protection in this study.

The immune response to HIV-1 virus-like particles (VLPs), presenting a clade A Ugandan gp120, has been evaluated in a mouse model by intra-nasal (i.n.) administration by a VLP+VLP homologous or a DNA+VLP heterologous prime-boost immunization protocol, including a HIV-1 DNA gp160/rev plasmid. Furthermore, the effect of the Eurocine lipid-based mucosal L3 adjuvant on the VLP immunogenicity has been assessed as well. The designed heterologous protocol is able to increase the env-specific humoral and cellular immune response, compared to the homologous protocol, which is to some extent increased by the administration of L3-adjuvanted VLP boosting dose. The anti-gag response is statistically increased in both homologous and heterologous protocols, particularly when the VLP boosting dose is adjuvanted. Immune sera from immunized animals exhibit >50% ex vivo neutralizing activity against heterologous A and B-clade viral isolates. An envelope B-cell epitope mapping shows an enhanced response against V3 epitopes all across the C2-V5 region in the heterologous prime-boost immunization strategy. The induction of humoral immunity at mucosal sites, which represents the main port of entry for the HIV-1 infection, is extremely relevant. In this framework, the DNA-VLP heterologous prime-boost protocol appears a promising preventive vaccine approach which can significantly benefit from specific mucosal adjuvants, as the Eurocine L3.

More focused research on a mucosal HIV-1 vaccine is needed urgently. An increasing amount of collected data, using heterologous multimodality prime-booster strategies, suggest that an efficient and protective HIV-1 vaccine must generate broad, long-lasting HIV-specific CD8(+) cytotoxic T-lymphocyte and neutralizing antibody responses. In the mucosa, these responses would be most effective if a preferential stimulus of HIV-1 neutralizing secretory immunoglobulin A and G were obtained. The attractive property of mucosal immunization is the obtained mucosal and systemic immunity, whereas systemic immunization induces a more limited immunity, predominantly in systemic sites. These objectives will require new vaccine regimens, such as multiclade HIV DNA and protein vaccines (nef, tat, gag and env expressed in DNA plasmids) delivered onto mucosal surfaces with needle-free delivery methods, such as nasal drop, as well as oral and rectal/vaginal delivery, and should merit clinical trials.

Objective: To test the efficacy of an immunoconjugate against HIV-1., Design: : A murine monoclonal antibody against the envelope antigen of HIV (P4/D10) was conjugated with the conventional anticancer drug, doxorubicin, and tested against infectious virus and infected cells, both in vitro and in vivo., Methods: P4/D10 antibody was incubated with free virus (neutralization) or HIV-infected cells (inhibition) and the resulting infection was measured by a p24 capture enzyme-linked immunosorbent assay. In an HIV-1/MuLV murine challenge model, the ability of the conjugate to inhibit infection in vivo was measured., Results: Doxorubicin-conjugated P4/D10 neutralized HIV-1IIIB and eliminated intercellular spread and HIV replication in infected Jurkat cells in vitro. The conjugate also protected mice from challenge with HIV-1IIIB/MuLV at an eightfold lower concentration than needed for free antibody, whereas no effects were observed for comparable doses of free drug or irrelevant conjugate controls., Conclusion: This indicates that doxorubicin is concentrated to HIV-infected cells by the P4/D10 antibody, significantly (P = 0.0001) contributing to HIV elimination. This concept could also be adapted to eradicate remaining antigen-expressing T cells in patients treated with antiretroviral therapy.

To determine the ability of antiretroviral treatment (ART) to deter viral replication in the long term, we tested autologous neutralization of HIV-1 primary isolates in sera from six chronically HIV-1-infected patients before and during such treatment. For comparison, heterologous neutralization was tested in the same samples by using a panel of eight primary HIV-1 isolates. Preceding ART, none of the patients' samples contained neutralizing antibodies against autologous HIV-1 isolates. Subsequently, despite successful ART treatment during a 12- to 19-month period and a rise in CD4 T cell counts, the patients' viral neutralizing capacity did not increase significantly. Furthermore, the partial heterologous neutralizing response was not improved in these patients. This outcome signifies the failure of an HIV-1-specific humoral immune response to improve, despite successful ART. Therefore, our results emphasize the need for immunologic intervention before cessation of ART in chronically HIV-1-infected patients to achieve sustainable control of viral replication.

Aims: The study was designed to evaluate a novel cationic lipid DNA adjuvant (N3) and its function for HIV-1gp160/rev DNA plasmid delivered intranasally. The primary N3/HIV-DNA plasmid immunizations were boosted intranasally with a gp41 peptide in a anionic L3 adjuvant. This novel prime-boost strategy of mucosal immunization provided a broad HIV-1 envelope specific immunity, and recognition of viruses of subtypes A, B and C., Conclusions: Intranasal N3-adjuvanted gp160/rev DNA prime followed by one L3-peptide boosting immunization, induced broadly neutralizing antibodies against HIV-1 in the mucosa and systemically. The needle-free intranasal prime-boost strategy using two different adjuvant formulations reduced significantly the dose of DNA needed.

We investigated the effects of immunizing with several genes and subtypes of HIV-1. The genes used as immunogens were: gp160 envelope (env subtypes A, B and C), p37gag (gag subtypes A and B), rev (subtype B) and reverse transcriptase (RT subtype B). The different genes are all carried by separate plasmids. C57BL/6 and BALB/c mice were immunized with different combinations of the genes together with recombinant cytokine granulocyte macrophage-colony stimulating factor. The env genes injected alone induced significantly stronger cellular responses to envelope in both strains of mice than when env genes were injected together with gag and RT genes. In the C57BL/6 mice, the envelope specific responses were significantly increased after spatial separation of env genes from gag and RT genes as compared to when all vaccine genes were injected as a mixture. The gag responses were strong in gag-immunized animals and were not significantly affected by the spatial separation of gag and RT genes from the env genes. Our results illustrate the importance of being cautious when formulating multivalent genetic vaccines and that it might be possible to overcome lost immune responses through spatial separation of vaccine antigens.

Background: The human FUT2 gene encodes the alpha(1,2)fucosyltransferase that determines secretor status. Homozygous for the nonsense mutation are called non-secretors and are unable to express histo-blood group antigens in secretions and on mucosal surfaces. In this study we have investigated the importance of the FUT2 fucosyltransferase activity on the progress of HIV-1 infection., Methods: Swedish blood donors (n = 276), 15 long-term non-progressors (LTNP) and 19 progressors were genotyped with respect to the nonsense mutation 428G-->A in the FUT2 gene. In addition 265/276 blood donors and 19 progressors with rapid or expected progression rate were Delta32 CCR5 genotyped., Results: Of 276 blood donors 218 (79%) were found to be secretor positive (se+), either homozygous (se+/+) wild type (30%) or heterozygous (se+/-) (49%) and 58 (21%) were homozygous non-secretors (se-/-). Five LTNP (33%) were found to be secretor-positive (se+/+, se+/-) and 10 (67%) se-/-. Of the 19 individuals with normal HIV-1 progression 15 (79 %) were found to be secretor positive and four (21%) were non-secretors. No frequency differences were found in the Delta32 CCR5 allele among the groups studied., Conclusion: Strong association (P < 0.001) was observed between the nonsense mutation 428G-->A in the FUT2 gene and a slow disease progression of HIV-1 infection.

The impaired functional activity of cytotoxic T lymphocytes and natural killer cells during HIV-1 infection has recently been attributed to decreased intracellular levels of perforin and granzyme B. In sera from individuals chronically infected with HIV-1 we report increased levels of extracellular perforin compared with uninfected individuals. Increased perforin was also observed during experimental SIV/SHIV infection. The combination of reduced intracellular perforin levels and an increased serum level indicates that HIV infection induces aberrant perforin secretion.

Gene vaccination encounters problems different from those of gene therapy since both a short half-life of the gene and a strong immune response to the gene product are desirable. We have evaluated a DNA vaccine consisting of seven plasmids encoding nine HIV-1 proteins. Using a needle-free delivery device, the Biojector, together with recombinant mouse GM-CSF, this vaccine induced strong gp160 Env- and p24 Gag-specific cellular and humoral immune responses in mice. The rGM-CSF was crucial for inducing both antibodies and antigen-specific CD8(+) T cell responses against both gp160 and p24. A GMP-produced lot of this vaccine, intended for human use, was delivered intradermally or intramuscularly into BALB/c mice at a GLP-accredited animal facility. This vaccine induced strong cellular responses independent of the route of immunization; moreover, no signs of toxicity were detected after histopathological examination of various tissues. Overall, the results indicate that the intradermal delivery of multigene/multisubtype HIV DNA in combination with recombinant GM-CSF is a safe and efficacious strategy for inducing high levels of specific CD8(+) T cells and unusually high titers of antibodies. This vaccine has been approved by the Swedish Medicinal Products Agency and is currently in a Phase I clinical trial.

The viral diversity of HIV-1 is likely to require a vaccine strategy that induces broad cellular and humoral anti-HIV-1 immunity. Our strategy is based on multiple HIV-1 DNA immunogens together with adjuvant recombinant granulocyte-macrophage stimulating factor. This article describes pre-clinical and clinical work preceding the initiation of clinical HIV-1 phase I/II trials.

The correlate of protection in human immunodeficiency virus (HIV) infection is not known, but preclinical and clinical studies support the involvement of both antibodies and cellular immunity. In addition, the existence of multiple HIV clades makes HIV vaccine design especially challenging. We have constructed a vaccine platform with an HIV-1 subtype B DNA immunogen expressing full length consensus sequences from HIV-1 rev, nef, tat, and gag with additional cellular epitope clusters from the env and pol regions. Furthermore, this platform has been extended to three additional plasmids expressing the same immunogens but originating from subtypes A or C consensus or FGH ancestral sequences. Immunogenicity in BALB/c mice, by gene gun or intramuscular delivery, revealed strong IFN-gamma production in response to in vitro re-stimulation with a H-2d restricted gag peptide (AMQMLKETI) or even stronger toward an env epitope (RGPGRAFVTI). Weak humoral immunity was detected. Gene gun immunization with a cocktail of all four plasmids induced pre-challenge cellular immunity in C57Bl6/A2.01 mice and subsequently a robust frequency of protection (11/12 animals) after experimental challenge with subtype A or B HIV-1/Murine Leukemia Virus (HIV-1/MuLV). The cross-clade protection observed in this challenge experiment demonstrates that these multigene/multiepitope HIV DNA immunogens are likely to be potent immunogens also against the HIV-infection of human beings.

An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1-9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse's life span.

Immortalized and transduced cell lines are traditionally used in model of the HIV-1 life cycle. Primary cells may better represent the tissue of origin and events in vivo. We utilized an HIV-1/murine leukemia A4070 pseudotype virus and human Cyclin T1 to replicate HIV-1 in primary murine cells, and demonstrate that primary murine cells support HIV-1 infection better than immortalized cells.

Drug resistance is becoming a problem in the treatment of the human immunodeficiency virus type one (HIV-1). To obtain therapeutic DNA vaccines that would target multiple drug-resistance (DR) mutations, we cloned genes for DR HIV-1 reverse transcriptase (RT) and codon-optimized synthetic genes encoding clusters of human CTL epitopes located at the sites of DR-mutations (RT minigenes) and antibody and CTL-epitope tags. Expression of RT genes/minigenes in eukaryotic cells was confirmed by Western blotting and immunofluoresence staining with RT- or tag-specific antibodies. Immunization of mice with DR-RT gene induced no RT-specific antibodies. Immunization of HLA-A(*)0201-transgenic mice with RT minigenes induced RT-specific cellular responses detected by interferon-gamma secretion. This documents first steps in creating therapeutic vaccine against drug-resistant HIV strains.

Superinfection by HIV-1 of a cell line containing the complete murine leukemia virus (MuLV) genome was shown to give rise to pseudotyped HIV-1/MuLV. Such superinfection was successful with certain strains of HIV-1 subtypes A-D. Primary spleen cells and cells of the peritoneal cavity of immunocompetent mice of the C57Bl/6 strain were infectable with the pseudotype HIV-1/MuLV and secreted HIV-1 in vitro and in vivo. In contrast, the murine cell lines, NIH 3T3, myeloma cell line Sp2/0, and two murine hybridoma cell lines were relatively resistant to infection and produced no or little HIV. After primary murine spleen cells had been infected with pseudotyped HIV-1 and transferred to C57Bl/6 mice, replication-competent HIV-1 was obtained from the peritoneal cavity for at least 10-14 days. High amounts (> 10(5) vRNA copies/ml) of HIV-1 vRNA could be measured in the peritoneal fluid. Presence of HIV-1 proviral DNA was detectable in cells from the peritoneal cavity for up to 24 days after infected cell transfer. Active reverse transcriptase representing both HIV-1 and C-type murine retroviruses was detected in the peritoneal washes. The HIV-infected spleen cells injected into the peritoneal cavity elicited HIV-1-specific cellular immune responses to p24gag, gp160Env, Nef, Tat and Rev. Mice immunized with HIV-1 DNA, but not with HIV-1 protein, cleared their HIV-1-infected cells within 10-14 days after challenge with HIV-1/MuLV-infected syngeneic spleen cells. This novel model system of primarily cellular reactivity to HIV-1-infected cells in vivo may become useful for assaying experimental HIV-1 immunization schedules., (Copyright 2004 S. Karger AG, Basel)

Ag-presenting dendritic cells present viral Ags to T cells after uptake of apoptotic bodies derived from virus-infected cells in vitro. However, it is unclear whether apoptotic virus-infected cells are capable of generating immunity in vivo. In this study, we show that inoculation of mice with apoptotic HIV-1/murine leukemia virus (MuLV)-infected cells induces HIV-1-specific immunity. Immunization with apoptotic HIV-1/MuLV-infected syngeneic splenocytes resulted in strong Nef-specific CD8(+) T cell proliferation and p24-induced CD4(+) and CD8(+) T cell proliferation as well as IFN-gamma production. In addition, systemic IgG and IgA as well as mucosa-associated IgA responses were generated. Moreover, mice vaccinated with apoptotic HIV-1/MuLV cells were protected against challenge with live HIV-1/MuLV-infected cells, whereas mice vaccinated with apoptotic noninfected or MuLV-infected splenocytes remained susceptible to HIV-1/MuLV. These data show that i.p. immunization with apoptotic HIV-1-infected cells induces high levels of HIV-1-specific systemic immunity, primes for mucosal immunity, and induces protection against challenge with live HIV-1-infected cells in mice. These findings may have implications for the development of therapeutic and prophylactic HIV-1 vaccines.

In a multisubtype approach to HIV-1 vaccination, mice were immunized with HIV-1 envelope gp160 genes from subtypes A, B, and C. Subsequently the mice were challenged with syngeneic primary splenocytes infected with a HIV-1/MuLV pseudovirus carrying a subtype B genome. HIV-specific immune responses and protection were strongest in the group of animals immunized with a combination of subtype A, B, and C specific gp160 genes as compared to subtype B only. Immunization with the combination of the cross-reactive subtypes A and C envelope genes induced HIV-specific immune responses but did not result in significant protection to challenge with subtype B infected cells. From this we conclude that immunization with the envelope genes from several HIV-1 subtypes may indeed enhance immune responses. This study shows that by using a mix of subtype envelope genes, an enhanced protective immunity can be obtained experimentally, potentially also in humans.

Background: HIV-specific IgA is present in HIV-exposed uninfected individuals (EU) and neutralizes primary strains of HIV-1 in vitro., Objectives: To analyse the antigenic correlates of HIV-1 neutralization using HIV epitopes and IgA from EU and HIV-seropositive individuals., Methods: Sera from six heterosexual couples discordant for HIV serostatus, six age-matched HIV-infected subjects and six healthy controls (HC; as negative controls) were analysed. IgA binding on HIV Env recombinant proteins was assayed. Serum IgA was affinity purified on specific Env peptides and tested in HIV neutralization using resting and activated peripheral blood mononuclear cells as target. Monoclonal antibody 2F5 was used as neutralizing positive control. BALB/c mice were immunized with specific gp41 peptide and anti-sera were tested in syncytia formation and in HIV viral replication., Results: IgA of EU exclusively bound an epitope within gp41; this epitope was restricted to residues 582-588 (QARILAV) and corresponded to the leucine zip motif in the alpha-helical region. IgA of HIV-positive patients recognized epitopes expressed both in gp120 and gp41; these epitopes were in the N-terminal portion of the extramembrane region. Additionally, IgA of EU and antisera of QARILAV-immunized Balb/C mice blocked syncytia formation and viral replication. The dose-dependent neutralization behaviour of specific QARILAV-purified IgA was very similar to that obtained with monoclonal antibody 2F5., Conclusion: These results have important implications for the development of vaccines and therapeutical strategies against HIV infection., (Copyright 2002 Lippincott Williams & Wilkins)

There is an urgent need for a universally effective HIV-1 vaccine, but whether a vaccine will be able to protect against HIV-1 of different clades is a significant concern. IgA from HIV-1-exposed, persistently seronegative (HEPS) subjects has been shown to neutralize HIV-1 and to block epithelial HIV-1 transcytosis, and it may target novel HIV-1 epitopes. We have tested the ability of plasma and mucosal IgA purified from HEPS subjects to neutralize HIV-1 primary isolates of different viral clades and phenotypes. IgA from two groups of HEPS subjects was tested: sex workers from Nairobi, Kenya, where clades A and D predominate, and the heterosexual partners of individuals infected by clade B virus. HIV-1-infected and low-risk uninfected individuals were included as controls. IgA purified from the blood, genital tract, and saliva of most HEPS sex workers demonstrated significant cross-clade HIV-1 neutralization, whereas a more clade-restricted pattern of neutralization was found in partners of clade B-infected individuals. IgA purified from HIV-1-infected individuals also mediated cross-clade neutralization, whereas IgA from uninfected controls lacked neutralizing activity. In conclusion, mucosal and plasma IgA from HEPS subjects neutralizes HIV-1 of different clades. This ability to induce HIV-1-specific systemic and mucosal IgA may be an important feature of an effective prophylactic HIV-1 vaccine.

Clinical and experimental studies of HIV-1 subcomponents were made in order to increase their immunogenicity. HIV subtype envelopes A, B and C have been compared and a detailed analysis made by peptides of the coreceptor-ligand interactions. We identified a direct interaction between HIV-1 envelope and a cellular receptor at the amino acid level. Both the viral subtype and its tropism appeared to influence inhibition of infection. Genetic immunization induced new cytotoxic responses while proteins appeared to efficiently boost previous responses. One HIV-1 subtype B antigen was strongly immunogenic in a human immunotherapeutic trial and permitted better survival at 2 years of the study in patients with poor prognosis.

DNA encoding HIV-1 env is a poorly efficient B-cell immunogen and one probable explanation is that the numerous gp120 N-linked glycans gp120 may interfere with B-cell epitope presentation. The N306 glycan in gp120 shields HIV-1 from neutralizing antibodies. A DNA immunogen lacking the N306 glycosylation signal (T308A) was constructed to determine whether this glycan affected the immune response. Mice were immunized intranasally twice with DNA containing either the wild type or the mutant env. Two additional groups were primed with wild type or mutant env and boosted with rgp160 protein, containing the complete set of N-linked glycans. Immunization with DNA alone resulted in priming of B-cell clones but was not sufficient to induce a complete antibody response. Animals primed with the N306 mutant and subsequently boosted with rgp160 protein displayed higher serum IgG-binding titers to gp120 than animals primed with wild type env DNA. The manipulation of the glycosylation sites of the env DNA strongly primes antibody responses (but non-neutralizing) as well as T-cell responses to the wild type strain gp160. However, priming with mutant plasmid did not result in higher neutralization titers to wild type or T308A-mutated virus than did the wild type plasmid. With the N306 mutant DNA we thus immunized a non-neutralization epitope, but obtained strong env-binding IgG after rgp160 boosting.

Although HIV-specific cellular immune responses are found in a number of HIV highly-exposed, persistently seronegative (HEPS) cohorts, late seroconversion can occur despite pre-existing cytotoxic T lymphocytes (CTL), suggesting that a protective HIV vaccine may need to induce a broader range of HIV-specific immune responses. Low levels of HIV-specific IgA have been found in the genital tract and plasma of the majority of Nairobi HEPS sex workers and appeared to be independent of HIV-specific cellular responses. IgA purified from genital tract, saliva and plasma of most HEPS sex workers were able to neutralize infection of PBMC by a primary (NSI) clade B HIV isolate, as well as viral isolates from clades A and D, which predominate in Kenya. In addition, these IgA were able to inhibit transcytosis of infective HIV virions across a transwell model of the human mucosal epithelium in an HIV-specific manner. Preliminary work in other HEPS cohorts has suggested the recognition of different gp41 epitopes in HEPS and HIV-infected subjects. Although present at low levels, these IgA demonstrated cross-clade neutralizing activity and were able to inhibit HIV mucosal transcytosis, suggesting an important functional role in protection against HIV infection.

DNA immunization permits evaluation of possible antagonistic or synergistic effects between the encoded components. The protein expression capacity in vitro was related to the immunogenicity in vivo of plasmids encoding the HIV-1 regulatory genes tat rev, and nef. Neither Tat nor Rev expression was influenced by co-expression in vitro of all three proteins, while Nef expression was slightly inhibited. With the combination of genes, the T-cellular responses of mice against Rev and Nef were inhibited compared with those when single gene immunization was used. No interference was detected for the Tat T-cell response. Thus, co-immunization with certain genes may result in inhibition of specific immune responses., (Copyright 2001 Academic Press.)

An inactivating mutation in the human CCR5 gene reduces the risk of HIV-1 infection in individuals with homozygous alleles. We explored whether genetic immunization would induce an immune response directed to CCR5 structures and if immunological tolerance toward endogenous CCR5 could be broken. We also studied whether this immunization approach could protect cynomolgus monkeys from an infection, with SIVsm, which primarily uses CCR5 as a coreceptor. Epidermal but not intramuscular delivery of the CCR5 gene to mice elicited strong IgG antibody binding responses to CCR5. Intramucosal immunization of cynomolgus macaques with CCR5 DNA followed by boosts with CCR5 peptides induced prominent IgG and IgA antibody responses in serum and vaginal washings. The CCR5-specific antibodies neutralized the infectivity of primary human R5 HIV-1 strains, and the macaque SIVsm but not that of a tissue culture-adapted X4 HIV-1 strain. The consecutive CCR5 gene and CCR5 peptide immunizations induced B- and T-cell responses to peptides representing both human and macaque amino acid sequences of the respective CCR5 proteins. This indicates that tolerance was broken against endogenous macaque CCR5, which has a 98% homology to the human CCR5 gene. After the final boost, the vaccinated monkeys together with two control monkeys were challenged with SIVsm. Neither protection against nor enhancement of SIVsm infection was achieved., (Copyright 2000 Academic Press.)

HIV-1-specific IgA has been described in the genital tract and plasma of HIV-1 highly exposed, persistently seronegative (HEPS) individuals, and IgA from these sites has been shown to neutralize HIV-1. This study examines the ability of IgA isolated from HEPS individuals to inhibit transcytosis across a tight epithelial cell layer. A Transwell system was established to model HIV-1 infection across the human mucosal epithelium. The apical-basolateral transcytosis of primary HIV-1 isolates across this mucosal model was examined in the presence and the absence of IgA isolated from the genital tract, saliva, and plasma of HEPS individuals enrolled in both a sex worker cohort in Nairobi, Kenya, and a discordant couple cohort in Italy. In the absence of IgA, HIV-1 primary isolates were actively transported across the epithelial membrane and were released on the opposite side of the barrier. These transcytosed HIV-1 particles retained their ability to infect human mononuclear cells. However, IgA purified from the mucosa and plasma of HEPS individuals was able to inhibit HIV-1 transcytosis. Inhibition was seen in three of six cervicovaginal fluid samples, five of 10 saliva samples, and three of six plasma samples against at least one of the two primary HIV-1 isolates tested. IgA from low risk, healthy control subjects had no inhibitory effect on HIV-1 transcytosis. The ability of mucosal and plasma IgA to inhibit HIV-1 transcytosis across the mucosal epithelium may represent an important mechanism for protection against the sexual acquisition of HIV-1 infection in HEPS individuals.

Objective: To characterize functional properties of HIV-specific IgA in samples representing both systemic and mucosal compartments of HIV-1 highly exposed persistently seronegative (HEPS) individuals., Methods: IgA was purified from plasma and mucosal samples from HEPS individuals and tested for the ability to neutralize infection of peripheral blood mononuclear cells (PBMC) by a non-syncytium inducing HIV-1 (clade B) primary isolate. None of these individuals had measurable HIV-1-specific IgG., Results: HIV-1-specific neutralizing activity of the purified IgA from plasma (n = 15), saliva (n = 15) and cervicovaginal fluid (CVF) (n = 14) were found in the majority of samples (73, 73 and 79%, respectively). In contrast, plasma, saliva and CVF samples of low-risk, uninfected HIV-seronegative individuals lacked neutralizing IgA, with the exception of two out of 34 (6%) saliva samples., Conclusion: Mucosal and plasma IgA from HEPS individuals can neutralize HIV-1 infection.

We investigated the immune response against a human immunodeficiency virus type 1 (HIV-1) nef DNA sequence administered epidermally in mice transgenic for the human major histocompatibility complex (MHC) class I molecule HLA-A201. Ten potential HLA-A2 binding 9-mer Nef peptides were identified by a computer-based search algorithm. By a cell surface MHC class I stabilization assay, four peptides were scored as good binders, whereas two peptides bound weakly to HLA-A2. After DNA immunization, cytotoxic T lymphocyte (CTL) responses were predominantly directed against the Nef 44-52, 81-89, and 85-93 peptides. Interestingly, the 44-52 epitope resides outside the regions of Nef where previously described CTL epitopes are clustered. Dominance among Nef-derived peptides did not strictly correlate with HLA-A2 binding, in that only one of the high-affinity binding peptides was targeted in the CTL response. The 44-52, 85-93, and 139-147 peptides also generated specific CTLs in response to peptide immunization. T helper cell proliferation was detected after stimulation with 20-mer peptides in vitro. Three Nef regions (16-35, 106-125, and 166-185) dominated the T helper cell proliferation. The implications of these results for the development of DNA-based vaccines against HIV is discussed., (Copyright 2000 Academic Press.)

Immunization with a recombinant glycoprotein 160 envelope immunogen derived from a virus of genetic subtype B induced strong specific T-helper cell responses in asymptomatic human immunodeficiency virus (HIV) carriers infected with subtypes B to G. This indicates that the HIV-specific T-helper immunity, which is the basis for development of antibodies and cytotoxic T lymphocytes, can be improved by both homologous and heterologous antigens. It also suggests that a particular immunogen can be effective against many different HIV strains.

An HIV-1 p24 capture enzyme linked immunosorbent assay (ELISA) was developed and used in a study of B-cell epitopes in rabbits immunised with different gag p24 antigens. Rabbits were immunised with virion HIV-1/Lai, baculovirus recombinant p24, Escherichia coli recombinant p24-15 and a mixture of synthetic peptides representing sequences of HIV-1 gag p24 protein, respectively. Five out of nine rabbits developed antibodies that could be used for an antigen capture ELISA. No significant differences in IgG titers to the whole gag protein were seen when comparing rabbits immunised with four different antigens. Three major common linear epitope regions were mapped in the rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24. The rabbit immunised with HIV-1 gag peptides had the broadest linear epitope reactive responses whereas animals immunised with E. coli recombinant antigen had the most restricted linear epitope response. The capture ELISA method thus developed using the different rabbit anti-p24 IgG preparations was shown to capture isolates from HIV-1 subtypes or clades A to G. Only rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24 developed IgG that was capable of efficiently capturing HIV-1 p24 in ELISA, indicating the importance of preparing antibodies able to recognise native or discontinuous and linear antigen configurations.

An HIV-1-specific miniantibody, a peptide representing the third heavy chain complementarity-determining region (CDR) of an HIV-specific mouse antibody, was characterized and modified with unnatural D-isomeric amino acids. The CDR peptide and its parent antibody bound to a similar epitope, located in the V3 region of HIV-1 gp120. A shortened CDR sequence was modified with D-amino acids to create an all-D-amino acid retro-inverso (RI) peptide with a reversed sequence order. The RI CDR was less susceptible to proteolytic degradation than its L-counterpart and had a higher affinity for HIV-1 peptides. The miniantibody and its parent antibody showed neutralization of both primary and laboratory strains of HIV-1. In accordance with the binding studies, the RI CDR showed a stronger HIV-inhibiting capacity than its L-counterpart. We conclude that the anti-HIV retro-inverso CDR identified in this study has the potential to become a future anti-HIV drug. It has a virus-neutralizing capacity in vitro and appears to be stable. Future research should focus on characterizing its antiviral activity in vivo.