Codon optimization and improved delivery/immunization regimen enhance the immune response against wild-type and drug-resistant HIV-1 reverse transcriptase, preserving its Th2-polarity.

DNA vaccines require a considerable enhancement of immunogenicity. Here, we optimized a prototype DNA vaccine against drug-resistant HIV-1 based on a weak Th2-immunogen, HIV-1 reverse transcriptase (RT). We designed expression-optimized genes encoding inactivated wild-type and drug-resistant RTs (RT-DNAs) and introduced them into mice by intradermal injections followed by electroporation. RT-DNAs were administered as single or double primes with or without cyclic-di-GMP, or as a prime followed by boost with RT-DNA mixed with a luciferase-encoding plasmid ("surrogate challenge"). Repeated primes improved cellular responses and broadened epitope specificity. Addition of cyclic-di-GMP induced a transient increase in IFN-γ production. The strongest anti-RT immune response was achieved in a prime-boost protocol with electroporation by short 100V pulses done using penetrating electrodes. The RT-specific response, dominated by CD4+ T-cells, targeted epitopes at aa 199-220 and aa 528-543. Drug-resistance mutations disrupted the epitope at aa 205-220, while the CTL epitope at aa 202-210 was not affected. Overall, multiparametric optimization of RT strengthened its Th2- performance. A rapid loss of RT/luciferase-expressing cells in the surrogate challenge experiment revealed a lytic potential of anti-RT response. Such lytic CD4+ response would be beneficial for an HIV vaccine due to its comparative insensitivity to immune escape.

Immunization with HIV-1 envelope T20-encoding DNA vaccines elicits cross-clade neutralizing antibody responses.

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.

HIVIS-DNA or HIVISopt-DNA priming followed by CMDR vaccinia-based boosts induce both humoral and cellular murine immune responses to HIV.

BACKGROUND: In order to develop a more effective prophylactic HIV-1 vaccine it is important optimize the components, improve Envelope glycoprotein immunogenicity as well as to explore prime-boost immunization schedules. It is also valuable to include several HIV-1 subtype antigens representing the world-wide epidemic. METHODS: HIVIS-DNA plasmids which include Env genes of subtypes A, B and C together with Gag subtypes A and B and RTmut/Rev of subtype B were modified as follows: the Envelope sequences were shortened, codon optimized, provided with an FT4 sequence and an immunodominant region mutated. The reverse transcriptase (RT) gene was shortened to contain the most immunogenic N-terminal fragment and fused with an inactivated viral protease vPR gene. HIVISopt-DNA thus contains fewer plasmids but additional PR epitopes compared to the native HIVIS-DNA. DNA components were delivered intradermally to young Balb/c mice once, using a needle-free Biojector® immediately followed by dermal electroporation. Vaccinia-based MVA-CMDR boosts including Env gene E and Gag-RT genes A were delivered intramuscularly by needle, once or twice. RESULTS: Both HIVIS-DNA and HIVISopt-DNA primed humoral and cell mediated responses well. When boosted with heterologous MVA-CMDR (subtypes A and E) virus inhibitory neutralizing antibodies were obtained to HIV-1 subtypes A, B, C and AE. Both plasmid compositions boosted with MVA-CMDR generated HIV-1 specific cellular responses directed against HIV-1 Env, Gag and Pol, as measured by IFNγ ELISpot. It was shown that DNA priming augmented the vector MVA immunological boosting effects, the HIVISopt-DNA with a trend to improved (Env) neutralization, the HIVIS-DNA with a trend to better (Gag) cell mediated immune reponses. CONCLUSIONS: HIVIS-DNA was modified to obtain HIVISopt-DNA that had fewer plasmids, and additional epitopes. Even with one DNA prime followed by two MVA-CMDR boosts, humoral and cell-mediated immune responses were readily induced by priming with either DNA construct composition. Priming by HIV-DNA augmented neutralizing antibody responses revealed by boosting with the vaccinia-based heterologous sequences. Cellular and antibody responses covered selected strains representing HIV-1 subtypes A, B, C and CRF01_AE. We assume this is related to the inclusion of heterologous full genes in the vaccine schedule.

Predictive performance of telenursing complaints in influenza surveillance: a prospective cohort study in Sweden.

Syndromic data sources have been sought to improve the timely detection of increased influenza transmission. This study set out to examine the prospective performance of telenursing chief complaints in predicting influenza activity. Data from two influenza seasons (2007/08 and 2008/09) were collected in a Swedish county (population 427,000) to retrospectively determine which grouping of telenursing chief complaints had the largest correlation with influenza case rates. This grouping was prospectively evaluated in the three subsequent seasons. The best performing telenursing complaint grouping in the retrospective algorithm calibration was fever (child, adult) and syncope (r=0.66; p<0.001). In the prospective evaluation, the performance of 14-day predictions was acceptable for the part of the evaluation period including the 2009 influenza pandemic (area under the curve (AUC)=0.84; positive predictive value (PPV)=0.58), while it was strong (AUC=0.89; PPV=0.93) for the remaining evaluation period including only influenza winter seasons. We recommend the use of telenursing complaints for predicting winter influenza seasons. The method requires adjustments when used during pandemics.

Amount of maternal rotavirus-specific antibodies influence the outcome of rotavirus vaccination of newborn mice with virus-like particles.

In presence of low or high levels of rotavirus-specific maternal antibodies, the ability of newborn mice to respond to immunization with rotavirus RF 8*-2/6/7 VLPs, was evaluated. After parenteral vaccination, 100% of offspring born to low-antibody-titer dams developed rotavirus-specific IgG antibodies (n=7). In contrast, only 25% of offsprings born to high-antibody-titer dams responded to parenteral immunization (n=12). When comparing parenteral versus oral immunization in offspring to low-antibody-titer dams only 45% responded after oral immunization (n=6). In conclusion, the response to parenteral immunization was not hampered by the presence of low levels of maternal antibodies induced by a natural infection while oral immunization was impaired. However, high levels of maternal antibodies impaired the response to parenteral immunization.

DNA-VLP prime-boost intra-nasal immunization induces cellular and humoral anti-HIV-1 systemic and mucosal immunity with cross-clade neutralizing activity.

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.

Bone marrow dendritic cells internalize live RF-81 bovine rotavirus and rotavirus-like particles (RF 2/6-GFP-VLP and RF 8*2/6/7-VLP) but are only activated by live bovine rotavirus.

Dendritic cells (DC) represent the link between innate and adaptive immunity. They are classified as antigen-presenting cells (APC) and can initiate and modulate the immune response. To investigate the interaction with DCs, live RF-81 bovine rotavirus strain (RFV) and rotavirus-like particles (rota-VLP), RF 2/6-GFP-VLP and rota RF 8*2/6/7-VLP, were added in vitro to murine bone marrow-derived DCs (bmDCs). Live RFV, RF 2/6-GFP-VLP and RF 8*2/6/7-VLP all bound to bmDC and were internalized but only live RFV stimulated phenotypic maturation of the bmDCs as shown by the upregulation of the co-stimulatory molecule CD86. Even though bmDCs internalized RF 2/6-GFP-VLP and RF 8*2/6/7-VLP as efficiently as live RFV, these rota-VLP were not able to activate the cells. Supernatants derived from bmDC cultures treated with live RFV, RF 2/6-GFP-VLP or RF 8*2/6/7-VLP were examined for TNF-alpha production. At 6, 18 and 24 h post-infection, TNF-alpha concentrations were significantly increased in cultures treated with live RFV and rota-VLP compared with untreated cultures. In conclusion, this study showed that live RF-81 bovine rotavirus strain was internalized and induced bmDCs activation, whereas both RF 2/6-GFP-VLP and RF 8*2/6/7-VLP were internalized by bmDCs without triggering their activation.

The rationale behind a vaccine based on multiple HIV antigens.

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.

Enhanced cellular immunity and systemic control of SHIV infection by combined parenteral and mucosal administration of a DNA prime MVA boost vaccine regimen.

The immunogenicity and protective efficacy of a DNA and recombinant modified vaccinia Ankara (MVA) vaccine administered by two different routes were investigated. DNA expressing HIV-1 IIIB env, gag, RT, rev, tat and nef, and MVA expressing HIV-1 IIIB nef, tat and rev and simian immunodeficiency virus (SIV) macJ5 gag/pol and vaccinia HIV-1 env, were used as immunogens. Four cynomolgus macaques received DNA intramuscularly (i.m.) at month 0 and intrarectally (i.r.) and intra-orally (i.o.) at 2 months, followed by MVA i.m. at 4 months and i.r. and i.o. at 8 months. Another group of four monkeys received the same immunogens but only i.m. Overall, stronger cellular immune responses measured by ELISPOT and T-cell proliferation assay were detected in the group primed i.m. and boosted mucosally. Following homologous intravenous simian-human immunodeficiency virus (SHIV) challenge, one of eight vaccinated animals was completely protected. This monkey, immunized i.m. and i.r.+i.o., exhibited the highest levels of HIV Env, Nef and Tat antibodies, high HIV Tat cytotoxic T-lymphocyte activity and T-lymphocyte proliferative responses to HIV Env. Four weeks post-challenge none of the monkeys immunized i.m. and i.r.+i.o., and only two out of four animals immunized i.m., demonstrated detectable plasma viral RNA levels. In contrast, all eight control animals had demonstrable plasma viral RNA levels 4 weeks post-challenge. Thus, stronger cellular immune responses and reduction of challenge virus burden were demonstrated in animals immunized i.m. as well as mucosally, compared with animals immunized i.m. only. The breadth and magnitude of the induced immune responses correlated with protective efficacy.

Multi-subtype gp160 DNA immunization induces broadly neutralizing anti-HIV antibodies.

A highly desirable feature for an human immunodeficiency virus type 1 (HIV-1) vaccine is the ability to induce broadly reactive anti-envelope antibodies that can neutralize primary HIV-1 isolates. Two immunizations with an HIV-1 envelope-encoding plasmid together with recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) resulted in high antibody titers in mice. The antibody induction was further enhanced after immunization with genes encoding HIV-1 envelopes originating from subtypes A, B and C. The sera from these animals were able to neutralize A, B and C viral isolates, whereas the sera from animals immunized solely with subtype B DNA neutralized only subtype B virus. The combined DNA vaccine gave serum antibodies with broad recognition of HIV-1 envelope epitopes as determined by peptide mapping. Cell-mediated immunity was not compromised by the increased humoral immunity. This demonstrates the ability of multiple envelope genes to induce the desired antibody response against several subtypes. Moreover, it documents the ability of rGM-CSF to enhance the potency of such a vaccine when given simultaneously. The strategy may be useful for making an HIV vaccine more potent and broadly effective against strains of different clades.

Genetic immunization is augmented by murine polyomavirus VP1 pseudocapsids.

To improve immune responses induced by DNA immunization, murine polyomavirus major capsid protein (VP1) pseudocapsids were complexed with a DNA plasmid encoding the p37 (p24 and p17) nucleocapsid proteins of the human immunodeficiency virus type 1 (HIV-1). A 10-fold increase in antibody titer was noted in mice given DNA plasmid together with VP1 pseudocapsids in comparison to animals that received DNA plasmid alone. Cell mediated responses to HIV-1 p24 occurred, but were not significantly augmented by delivering the DNA as a VP1 complex. We have consequently for the first time shown a carrier/adjuvant effect of polyomavirus pseudocapsids that strongly increased the humoral immune response in DNA immunization.

HIV subtypes and recombination strains--strategies for induction of immune responses in man.

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.

Functional HIV-1 specific IgA antibodies in HIV-1 exposed, persistently IgG seronegative female sex workers.

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.

Enhanced immunogenicity of a human immunodeficiency virus type 1 env DNA vaccine by manipulating N-glycosylation signals. Effects of elimination of the V3 N306 glycan.

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.

Interactions of single and combined human immunodeficiency virus type 1 (HIV-1) DNA vaccines.

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.

Induction of immune responses and break of tolerance by DNA against the HIV-1 coreceptor CCR5 but no protection from SIVsm challenge.

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.

Mucosal and plasma IgA from HIV-1-exposed uninfected individuals inhibit HIV-1 transcytosis across human epithelial cells.

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.

Mucosal and plasma IgA from HIV-exposed seronegative individuals neutralize a primary HIV-1 isolate.

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.

Immunogenic properties of reverse transcriptase of HIV type 1 assessed by DNA and protein immunization of rabbits.

Genetic immunization may be one way to prime individuals for a subsequent broad anti-HIV-1 immune response. Reverse transcriptase of HIV-1 (RT) presents a selective target for attempts to arrest replication of HIV-1. Rabbits immunized with a plasmid carrying the gene for reverse transcriptase HIV-1 (RT DNA) developed potent antibody and cellular responses to the gene product. The immunogenic properties of RT DNA and recombinant reverse transcriptase were compared in rabbits. The specific immune responses were similar to those reported previously for HIV-1 infected humans. The array of B and T cell epitopes recognized in RT DNA-immunized rabbits was broader than in rabbits immunized with the recombinant RT. We localized seven novel B and T cell epitopes and concordance between B cell and helper T cell epitopes was observed. B cell epitopes of RT induced proliferation of peripheral blood mononuclear cells and were active as helper T cell epitopes. T cell-proliferative responses to the epitopes of RT preceded or paralleled the production of antibodies of the same specificity. Subdomains of reverse transcriptase involved in the enzymatic activity of RT were highly immunogenic. Anti-RT IgG partially inhibited reverse transcription in vitro.

Human immunodeficiency virus type 1 Nef epitopes recognized in HLA-A2 transgenic mice in response to DNA and peptide immunization.

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.