BACKGROUND
twenty eight years after the first cases were recognized, the hiv- <dig> pandemic continues to grow exponentially resulting in more than  <dig> million cases of individuals living with hiv worldwide.
constant virus replication in cd <dig> t lymphocytes initiates progressive immune defects and finally, after  <dig> to  <dig> years, results in acquired immunodeficiency syndrome  and death.
the course of the hiv infection has changed significantly with the development of new antiretroviral regimens that combine inhibitors of reverse transcription, virus protein cleavage, or even virus entry.
they reduce viral burden and immune damage caused by hiv  <cit>  but cannot fully eradicate the virus.
thus, lifelong therapy is expected to transform this otherwise lethal disease into a chronic, continuously treated infection by preventing the progression to aids.
however, severe drug-related adverse effects and the development of drug resistance limit their efficacy, and the drugs have not been affordable for the vast majority of patients worldwide.
because a therapeutic breakthrough that would soon eradicate hiv or limit side effects appears unlikely at present, additional therapeutic strategies continue to be relevant to the lasting prevention of aids onset.
a better characterization of the initial host immune response to hiv- <dig> infection may help to define protective immunity to hiv- <dig>  one such strategy might be to combine antiretroviral treatment with immune responses to hiv.
some immune control of hiv is evidenced by the temporal association of virus reduction and the emergence of hiv-specific t cells  <cit> , however in the absence of a pre-infection stimulus, anti-hiv neutralizing antibodies normally develop too late to play a key role during natural infections.
findings have suggested that cellular immunity is involved in the initial control of virus replication in primary hiv- <dig> infection and indicate a role for ctl in protective immunity to hiv- <dig> in vivo.
importantly, analyses of vaccination studies in nonhuman primate have indicated that single viral epitope-specific ctl responses may not be sufficient to block infection with pathogenic siv  <cit> .
in turn this suggests that the generation of broader responses that target multiple viral epitopes may be critical to the development of effective protection against aids.
thus, a recent alternative approach has involved the use of multiple hiv antigens and the inclusion of both structural and regulatory antigens  <cit> .
an indication that this approach can be more effective has recently been verified in that in animals, protection from heterologous shiv challenge was observed only with immunization using an aggregated, multiantigen subunit protein vaccine that incorporated the structural protein env and the regulatory proteins tat and nef  <cit> .
when individually immunized using the same antigens, animals were not protected.
additionally, studies demonstrated improved protection from aids and cd4+t-cell depletion in animals immunized with a combination of antigens  <cit> .
inherently, the microparticulate gas vesicle organelles produced by halobacterium sp.
nrc- <dig> have numerous characteristics which would be desirable in an antigen presenting system and thus potentially can have importance for applications in vaccine development.
the organelles are readily amenable to genetic engineering techniques that produce gas vesicles expressing on their surfaces the selected exogenous amino acid sequences, and the presence of surface borne peptide sequences has been assessed and verified in vivo  <cit> .
unlike most phage or animal virus-derived vaccine components, the gas vesicle structure does not contain nucleic acid.
as a result, with the proteinaceous gas vesicle system the potential effects associated with co-introduction of dna sequences, inherent in viral or phage based vaccines, can be completely avoided.
the gas vesicles are readily, rapidly and inexpensively isolated from the bacteria by lysis in low salt and the isolated nanoparticles are uniquely stable.
large-scale production of gas vesicles is simple since these organelles float upon release from the halobacteria and thus are easily harvested.
since they are non-toxic to animals and humans, gas vesicles should have utility for use as oral as well as parenteral delivery vehicles.
as initially demonstrated, gas vesicles from a mutant halophilic archaea sd  <dig>  can be engineered to display various peptides by insertion of exogenous pathogen dna sequences into a site engineered into the gvpc gene.
thus, expression system plasmids were generated in which sivsm gene segments were inserted into site "d" engineered within gvpc gene  <cit> .
the plasmid constructs were subsequently used to transform the vac-  mutant, halobacterium nrc- <dig> strain sd <dig> and the recombinant gas vesicles harvested and isolated as described in detail previously  <cit> .
these nanoparticles were then pre-tested for immunogenicity, antigenicity and the activation of immunologic memory  <cit> .
in the present study we examined sera from different times post-booster and post re-immunization to assess the antibody isotype elicited in vivo by populations of gas vesicles  each expressing a selected sivsm peptide sequence.
using an in vitro macrophage cell culture system we then tested for possible effects of gas vesicle uptake on cytokines produced by cultured macrophages through quantification of three  key examples: il- <dig>  il- <dig> and il- <dig>  finally, given the known, ultra stable nature of the gas vesicle nanoparticles  <cit>  we used in vitro culture in conjunction with  immunofluorescence microscopy and  western blot assessments to examine sivsm and gvpc molecular weight species as indicators of their degradation.
thus, the changing intracellular distribution patterns of gvpc positive protein and the incorporated sivsm peptide, as well as the molecular weight species of degradation products were specifically examined.
RESULTS
immunoglobulins  are glycoproteins  present in serum and tissue fluids that recognize and bind their cognate antigen.
they constitute the adaptive, humoral immune responses, are non invasively accessible for testing and their utility in vivo is in part a function of their specific isotype.
as a first step in the present studies, we therefore evaluated our archived serum to assess antibodies elicited by recombinant gas vesicles displaying an sivsm peptide inserts  as a function of immunization status and overall elapsed time post-immunization or booster .
isotyping of immune sera
based on results from the preliminary sera screenings, igg <dig>  igg2a and igm were selected for evaluation utilizing the southernbiotech isotyping kit as described in the methods.
the assay results are presented graphically in figure  <dig> which plots the isotypes detected at the optimum antigen specific antibody dilution.
this antigen specific approach allowed determining the predominant isotype elicited and present at different times post-immunizaton or booster.
as the histograms in panels of figure  <dig> show, with two exceptions , igg <dig> is the predominant isotype at  <dig>   <dig> and  <dig> weeks post second booster.
seventeen weeks later, the mice were re-immunized using aliquots of the same r-gv preparations and sera collected at  <dig> days and again at  <dig> weeks post re-immunization.
the isotyping results for samples from these time points, shown in figure  <dig>  confirm that for all the mice tested, igg <dig> remains the predominant isotype including the assessments of sera drawn at  <dig> weeks post re-immunization.
for sera of all animals, at the optimum antigen specific dilution, igg <dig> remained the predominant isotype and its signal was greater than that of the  <dig> day post re-immunization samples indicating the specific antibody responses continued to develop over time.
determination of key cytokines elicited from j774a.
<dig> macrophage cells
in recent years the expression "cytokine network" has become commonly accepted in biomedical research and that network now includes several hundred members.
using our in vitro monolayer system this study assessed the levels of three key cytokines as a function of r-gv uptake by macrophage cell monolayers.
the studies had two aims: first to evaluate whether uptake of gas vesicle particles by these monolayer cells could influence the detectible levels of cytokines in the surrounding media and second, to determine whether there were differential cytokine levels as a correlate of the specific sivsm peptide expressed by the r-gvs applied to the individual monolayer cells.
positive findings would add to a profile that characterizes peptide specific effects in vitro as well as to the potential utility of this nanoparticle delivery system.
there are known interactive influences for each cytokine and macrophages normally produce il- <dig>  il- <dig> and, il- <dig>  among others.
il- <dig> and il- <dig> have important roles in inducing th <dig> responses while il- <dig> can antagonize the generation of th <dig> subset of helper t cells and enhance proliferation of b cells.
further, macrophages are widely distributed in mammalian tissues and fluids.
therefore assessment utilized monolayers of j774a.
<dig> murine macrophages and the protocols for in vitro r-gv application, followed by quantification of cytokines in the culture media as described in the methods section.
to test whether there might be r-gv insert specific differences, cell monolayers were incubated for  <dig> - <dig> h with wt-gv  a control, or with the different r-gvtat, rev or nef <dig>  at the times specified after gv removal and rinsing, aliquots of culture media were harvested and stored at -20°c until assessed for their content of the selected cytokines.
cytokine quantifications used point to point curves generated by assay of the individual purified cytokines provided in the cytokine kits and the calculation methods recommended by the kit manufacturer.
concentrations of the three different cytokines were assayed in media of cultured cells receiving no gas vesicles, media from duplicate cultures treated with one of the different sivsm insert containing r-gv populations, or with the insert negative wild type control from nrc- <dig>  table  <dig> presents these results which are snapshots in time of individual cytokine levels with the elevated levels shown in bold and shows there are notable differences in concentrations, and these vary as a function of the sivsm insert and also the time post application.
thus for r-gvtat or revsamples, concentrations of il- <dig> and il- <dig> were maximal at  <dig> h post application , then declined.
for samples of r-gvnef <dig>  the peak il- <dig> concentration was much higher and occurred at  <dig> h ; although lower in the  <dig> h samples, it was still elevated .
for il- <dig>  maximal concentration again occurred early at  <dig> h for r-gvtat treated cultures , then decreased to the level of the insert negative nrc- <dig> in the  <dig> h sample.
in contrast, for r-gvrev or nef <dig> media, increased pg/ml were detected only in the  <dig> h samples with nef <dig> exhibiting the higher concentration .
finally for il- <dig>  samples from r-gvtat or rev treated cultures, the  <dig> h samples displayed some elevation in concentration  that somewhat exceeds the elevated levels detected in samples from the cultures exposed to the insert negative control, gas vesicles from nrc- <dig> .
at later times, il- <dig> levels were additionally elevated by r-gvtat, with the higher levels occurring respectively at  <dig> and  <dig> h. .
in r-gvrev treated samples, the  <dig> h time displayed elevated il- <dig>  but this decreased to ~half that concentration in the  <dig> h and  <dig> h post-addition samples.
in contrast, at the different times tested, monolayers treated with r-gvnef <dig> displayed no significant increases in il- <dig> concentrations .
in fact the levels were marginally lower than those from samples from insert negative nrc- <dig> cultures .
therefore these assays indicate that unlike assays for r-gvtat and rev, r-gvnef <dig> elicited no significant insert specific increase in il- <dig> concentrations.
† as detailed in the methods, macrophage monolayers received media only.
‡ control wells received the wild type gas vesicles nrc- <dig>
although these data are "snapshots", nevertheless they indicate there are quantifiable, sivsm insert specific differences and time dependent effects on cytokines produced by j774a.
<dig> cell following the application and internalization of r-gvtat, rev or nef <dig>  thus in the context of an antigen delivery vehicle, there clearly is a detectible, insert specific impact on cytokine production by recipient monolayers.
these results therefore add to an overall picture characterizing the immunogen as delivered via the r-gv system.
they also suggest r-gvs could potentially be further designed for a co-delivery specifically aimed at simultaneously affecting cytokine levels.
intracellular fate of gas vesicles
at 50- <dig> nm, individual gas vesicle nanoparticles are below the limit of light microscope resolution  <cit>  but clusters of them, and of their breakdown products are detectible by immunostaining.
gas vesicles are remarkably stable particles  <cit>  and thus may inherently result in a slow release of the incorporated peptide sequences.
therefore r-gv, their fate and susceptibility to timely degradation constitute critical parameters in context of in vivo applications.
immuno-microscopy assessment of r-gv intracellular fate
studies directed at examining the post-phagocytic fate of the recombinant nanoparticles used specific antibody probes to detect the sivsm encoded peptides and the gvpc protein per se.
this protein contains the insert and specifically identifies gas vesicle particles.
using the protocols specified in the methods, the j774a.
<dig> macrophage cell line monolayers were prepared and incubated with r-gv then fixed as described at the different, specified times and stored at -20°c for subsequent assessments.
immunostaining with appropriate primary antibody allows immunofluorescent detection of r-gv delivered sivsm peptides -tat, rev or nef <dig> and separately, of the gas vesicle protein, gvpc.
the initial distribution of r-gv material following internalization by monolayer cells is shown by the low magnification  micrograph in figure  <dig>  it provides an overview of representative control and r-gvtat, rev and nef <dig> treated monolayers.
these samples were harvested at  <dig> h post r-gv treatment and subsequently immunostained with the anti-shiv monkey plasma which identifies the sivsm peptide.
use of this plasma verifies that antibodies elicited during in vivo infection with live, native shiv recognizes the r-gvsivsm peptides synthesized by the halobacteria as if they were native siv protein.
the monolayer in panel a of figure  <dig> is a control to test for non specific antibody binding.
it was not incubated with gas vesicle preparation and clearly the monolayer exhibits only a background fluorescence signal.
in contrast, monolayers treated with r-gv show numerous cell associated fluorescence positive regions in tat and rev treated monolayers .
for the larger nef <dig> insert , there also are cells that appear to "glow".
this fluorescence labelling shows the more extensive binding of anti-shiv primary antibody to the larger nef <dig> peptide contained in r-gvnef <dig> particles.
a second, critical characteristic is the susceptibility of the r-gv delivery vehicle to intracellular degradation that releases the siv inserts for processing by the cell.
the fate of the sivsm insert was followed in immunostained samples harvested over a period of 12- <dig> h and representative  <dig>  × images of immounostain results are presented in figure  <dig>  as would be anticipated, these show that the sivsm specific staining appears to become more diffusely distributed in the cells both as a function of the elapsed time, and the specific insert.
at  <dig> h, shiv positive particles  appear to be associated with the monolayer cells, internalized by them, often as a cluster  or internalized to produce a somewhat more diffuse green fluorescence .
by  <dig> h the bright punctuate staining of rev and tat has diminished, becoming more diffuse, and these cells do not 'glow'.
however for nef <dig>  the largest of the sivsm encoded insert tested, distinct regions of 'glow' remain, likely because the degradation of this larger siv insert remains less complete.
however by  <dig> h, the immunostaining for nef <dig> also has become more generalized.
interestingly, for the sample receiving r-gvrev, nuclear regions of the cells immunostain quite brightly and in the  <dig> h rev sample, this apparent nuclear association is clearly evident.
though not as distinct, a similar nuclear association occurs with the tat insert.
in contrast, the r-gvnef <dig> treated sample does not exhibit this distribution.
additionally, at the  <dig> h time, the diffuse, lightly stained areas detected in the cytoplasm of the r-gvnef <dig> sample appear similar to the cytoplasmic distributions detectible in the  <dig> h r-gvtat sample.
this later appearance in the r-gvnef <dig> treated monolayer might be a function of slower breakdown of this larger expressed peptide.
as expected, for each of the three different r-gv populations, the immunostaining of the incorporated sivsm derived inserts is consistent with ongoing r-gv breakdown of the individual clusters and concomitant disappearance of detectible, brightly fluorescent particle clusters.
other companion monolayers were similarly rinsed, fixed, then immunostained using anti-gvpc specific antibody to detect the larger, gas vesicle specific "c" protein and figure  <dig> presents a temporal series of representative images from 12- <dig> h post r-gv application.
relative to the sivsm inserts, gvpc is a large protein and like gas vesicle proteins in general, is resistant to dissociation/degradation  <cit> .
immunostaining to identify gvpc initially gives rise to cells exhibiting an overall glow that becomes significantly diminished by the  <dig> h time point and this would be consistent with a slow degradation of gvpc.
however, positive but muted and somewhat diffuse punctuate immunostaining remains at the  <dig> h time and can support suggesting that full degradation of gvpc remains incomplete at this time.
in the context of a display/delivery particle, it is important that cells can both internalize the r-gvs, and successfully degrade them, allowing release of the pathogen specific insert they carry.
the observed level and distribution of immuno-reactive gvpc material in monolayers at the  <dig> h time is fully consistent with ongoing particle degradation.
additionally, the decreasing brightness of the immunostaining supports suggesting the degradation time frame for and perturbation by gvpc will extend beyond the time required for the degradation of sivsm inserts.
western blot assessments verify intracellular breakdown of sivsm and gvpc components of r-gv
ultimately, the processing and removal of immunogens and their carriers is a significant factor for in vivo applications.
therefore the fate of the gvpc and sivsm components detected by immuno fluorescence microscopy during the late stages post r-gv application were examined further.
based on the patterns observed in these preparations, samples from the  <dig> h and  <dig> h were additionally assessed to verify protein breakdown.
other j774a.
<dig> monolayers were incubated with the gas vesicle preparations under the same conditions, then rinsed and harvested.
these samples were assessed by preparing cell lysates and separating the protein species using 4-12% sds-page gradient gels as detailed in the methods.
in acrylamide gels, pure intact gvpc protein behaves anomalously relative to its actual molecular weight, migrating as a species of ~ <dig> kda  <cit> .
following separation using sds-page, and transfer to pvdf membrane, gvpc peptides and the expressed sivsm insert were detected using the same anti-gvpc antibody stock and anti-shivsm monkey plasma utilized for the monolayers in immunostaining studies; companion halves of blot membranes were probed with the respective specific antibodies.
figure  <dig> presents typical western blots of samples from the  <dig> h  and  <dig> h  time points, probed respectively with anti-gvpc antibody or anti-shivsm plasma.
gvpc is synthesized by the recombinant bacteria and an integral, surface displayed component of these gas vesicles.
the observed immunostained bands of differing kda are a reflection of the molecular weights of gvpc breakdown products; other associated degradation resistant gas vesicle structural proteins also would be expected and if still integrated with gvpc, would affect its migration in gels  <cit> .
the presence of multiple protein bands identified for r-gv samples and for the shiv peptide is consistent with the immunostain microscopic observations which also suggested r-gv break down is a process that continues over a period of days.
the membrane half probed with anti-gvpc antibody specifically identifies gvpc protein and peptides.
it detected a number of bands at a variety of molecular weights, rather than the single ~ <dig> kda band that would characterize intact gvpc protein.
the majority of bands in blots of the  <dig>  and  <dig> h samples appear at < ~ <dig> kda with heavily stained bands detected at ~ <dig> kda and at somewhat at less than  <dig> kda.
the immunostain of these bands is notably stronger at the  <dig> h time, indicating gvpc breakdown is continuing and gives rise to these species, but clearly is still incomplete.
importantly, some bands may exhibit anomalous migration due to gas vesicle protein resistance to the standard protein dissociation protocols  <cit> .
slow disassembly and intracellular degradation would not be unexpected for gas vesicle proteins and likely perturbation of cells by partially degraded gvpc fragments would continue for an additional, but finite period of time.
in contrast, the companion sivsm inserts of rev, tat and nef <dig> remain detectible but for the  <dig> h sample, the peptides immunostain lightly with the anti-shiv antibody.
notably the inserts are an integral part of gvpc and like other gv proteins, gvpc is resistant to degradation.
multiple but light staining bands detected with the anti shiv antibody would therefore be expected.
at  <dig> h, the bands recognized by anti-shiv antibody are minimally in evidence.
no immunoreactive high molecular weight bands are obvious and the molecular weight species of ~ <dig> kda or less now are only minimally detectible.
clearly therefore, despite their integration into the original gvpc framework protein, the sivsm inserts are accessible to degradative processing.
therefore these foreign proteins ultimately should be cleared by cells.
discussion
the studies presented above were designed to further assess practical aspects of the recombinant gas vesicle display/delivery system through in vitro and in vivo studies.
as a self-adjuvanting, highly stable component  <cit> , r-gv have significant potential as an alternative delivery system and thus the studies reported were designed to provide additional characterizations.
the siv fragments tested were selected because they are key immunogens examined as relevant gene products in other published research  <cit>  those studies had shown toxoids of tat or tat and rev, or delivery of aggregated multiantigens, provided some disease attenuation  <cit>  whereas use of tat alone for example, provided only temporary infection containment.
this was interpreted to mean a single antigen might not be adequate to provide full and/or long term protection  <cit> .
our recombinant system inherently supports the expression and delivery of various epitopes  <cit>  and could be readily adapted if important, new peptides were identified.
thus combinations of r-gv particles could be easily created for use in vivo and this would provide exposure to multiple antigen fragments and thereby elicit a variety of peptide specific antibodies.
different isotypes bind to cell surface expressed fc receptors in vivo and the resulting receptor derived cell stimulation then will initiate a cascade of effects that can impact the local milieu, and ultimately cell functions.
utilizing the optimum antigen specific dilution of each antibody sample as determined by pre-titration using the specific tat, rev, and nef <dig> antigens, the present studies identified the isotype elicited by r-gv immunizations.
these analyses showed igg  was the predominant immunoglobulin species .
although for the r <dig> and n <dig> groups, igm was predominant at  <dig> days, at  <dig> weeks, in the absence of any subsequent booster, igg <dig> was the dominant isotype in all samples.
this can be relevant in vivo since antibody isotypes impact downstream events through antibody binding to cell displayed fc receptors  <cit> .
additionally, the continued presence of antigen specific mouse antibodies  <dig> days  after the final re-immunization, is potentially an important feature for an immunizing agent and likely to be relevant in terms of practical utility.
the isotyping data also inherently demonstrated that the peptides encoded by the expressed small dna inserts , are immunogenic and as shown in figure  <dig>  relative to the expressed nef <dig> encoded insert  appear to be degraded more slowly than their actual size would predict.
this finding may reflect the central location of these encoded small peptide inserts,  <dig> and  <dig> amino acids respectively, within the gvpc protein.
at that site they may be less readily accessible to the intracellular degradation processes.
nevertheless they are immune visible and evoke specific antibody responses.
gas vesicles are particulate moieties and when phagocytosed can impact various cell functions, including cytokine production.
in turn, both individually and in combination, cytokine levels significantly affect the nature and level of immune responses and response polarization in terms of antibody , and cell mediated immunity   <cit> .
in vivo, cytokines impact immune related processes and certainly specific roles for different cytokines have been identified and extensively investigated.
studies of hiv for example, have demonstrated cytokine correlations with protection from infection, prediction of disease progression and impact on immune response polarization  <cit> .
based on the potential for an insert specific effect, the present in vitro studies assessed the r-gv immunogens for their possible impact on selected cytokines, quantified by testing the media of monolayers treated with r-gvtat.rev and nef <dig>  vs. wt-gv, or left untreated.
although the siv specific peptide inserts constitute a very small segment in the recombinant gvpc protein, nevertheless in vitro studies showed there were detectible, insert specific differences in the cytokine patterns and the temporal profiles of their changing concentrations .
given the known effects of cytokines il- <dig>  il- <dig> and il- <dig>  and data in the literature relating to tat, ref and nef <dig>  the present findings show that despite the preponderance of gvpc specific protein, even very small segments of these pathogen proteins could play specific and significant roles in vivo by influencing the local milieu  <cit> .
although in vitro effects can not fully reflect in vivo process or responses, they nevertheless can indicate the potential to exert an effect.
analogous consequences in vivo could alter the local milieu and thereby alter the th1/th <dig> responses equation.
in turn, this might affect vaccine efficacy and certainly adds to a characterization of this immunization vehicle.
thus similar examinations would be relevant in the broader context of vaccine development, and of characterizing pathogen impact on tissues or systems.
known examples are inhibitory effects of il- <dig> on hiv- <dig> production in monocytes/macrophages resulting from il- <dig> stimulated inhibition of cytokine syntheses e.g.
tumor necrosis factor alpha  and il- <dig>  in the context of hiv- <dig>  this alteration can up-regulate pathogen expression in the infected cells  <cit> .
additionally, studies by others also have described il- <dig> inhibitory effects on cytokine synthesis  <cit> , and on pathogen  replication in monocytes  <cit> .
the inherent physical stability of the gas vesicle structure, along with its resistance to chemical dissociation or proteolytic cleavage  <cit>  and its particulate structure, likely play a role in the natural slow release of the incorporated epitope containing fusion proteins and could enhance stimulation of the immune system.
ultimately however, the full degradation of the particulate r-gv immunogen is important.
their long term presence potentially could elicit chronic, local inflammatory responses that can characterize degradation resistant particles.
the in vitro immunostaining and the western blot findings presented here were aimed at assessing intracellular gv breakdown.
the results indicate that degradation of these naturally stable immunogenic particles does occur and demonstrates the sizes of degradation products.
in this context, it is noteworthy that even long term, oral ingestion of gas vesicles on a regular basis  <cit>  has provided no evidence that gas vesicles exhibit any detectible short or long term toxicity.
thus per-oral usage of r-gv also could be an option.
micrographs from the immunofluorescence staining allow the tracking of r-gv intracellular fate after the application to j774a.
<dig> cells monolayers in vitro.
these studies demonstrated that as expected, r-gv are phagocytosed.
microscope focusing indicated r-gv associated with and/or were within j774a.
<dig> macrophages at  <dig> h post application and clearly internalized at later times .
results from the subsequent immunostain monitoring of both the gas vesicle protein gvpc, and the siv specific inserts confirmed their slow, progressive disappearance over time.
an intracellular accumulation/coalescence of individually internalized r-gv particles clearly takes place and is evident in the micrographs of  <dig> h post application monolayers.
additionally, samples from various times  following removal and rinsing off of the applied r-gvtat, rev or nef <dig> were immunostained using either anti- shiv or anti-gvpc antibody  and examined.
these clearly show a decrease in detectible immunostained material during this time course.
the results from this group of studies therefore provides critical information in terms of potential r-gv utility as a delivery vehicle since it indicates that in vivo there should be an ongoing but distinctly finite host cell perturbation following cellular uptake.
this likely would impact immune stimulation, potentially by processes including the stimulation of cytokine production detected and quantified above.
importantly the present studies tracking degradation indicated that over time in vitro, the recombinant particles did undergo degradation in the individual cells.
this was evidenced by the immunomicroscopy data for samples  and was verified in more detail by the western blot assessments which tested lysates of r-gv treated monolayer at  <dig> and  <dig> h post r-gv applications .
since recombinant inserts are expressed in gas vesicle protein 'c', studies assessed breakdown of this protein species and also of the specific expressed sivsm inserts.
in sds-page gels isolated, intact gvpc would appear as a single, ~ <dig> kda protein species  <cit> .
the western blot analysis using anti-gvpc showed clearly that wt-gv and the r-gvtat, rev and nef <dig> recombinants all exhibited multiple molecular weight species at both  <dig> and the  <dig> h times after application to monolayer cells.
this is the predicted result when proteins that become internalized are broken down and is consistent with the immunostaining detected in figures  <dig> and  <dig>  it also would be expected that the band patterns in sds-page gels, and the density of western blot immunostained bands would change as a function of time and this is the case .
thus, by  <dig> h, there are crisp gvpc positive bands of lower molecular weight species , as well as higher molecular weight species which are now more lightly stained by the anti-gvpc antibody.
all of these bands are reactive with the anti-gvpc antibody, and since in sds-page gels, gvpc that is un-degraded behaves as a  <dig> kda moiety  <cit> ; these clearly are degradation products from the gvpc protein.
they exhibit discrete, decreasing molecular weights consistent with degradation that is ongoing over time.
similarly the companion western blots probed with anti-shiv specific antibody demonstrated breakdown of the sivsm derived inserts and showed the disappearance of bands over time .
in addition to the detailed findings, the results overall support the conclusion that intracellular r-gv are susceptible to degradation and therefore can be cleared from cells that may phagocytose them.
furthermore, unlike the effects on cytokine concentrations, inserts in r-gvtat, rev or nef <dig> do not appear to significantly alter the r-gv time course of degradation in an insert dependant way.
CONCLUSIONS
the studies presented here provide an initial examination of recombinant gas vesicles  in terms of predominant antibody isotype elicited in vivo, particle impact on selected cytokines elicited following uptake by monolayer cells in vitro and r-gv internalization and intracellular degradation during in vitro culture.
the murine immunizations showed that despite the relatively small size of the expressed recombinant insert, like other haptens sivsm derived inserts incorporated into the gvpc "carrier" protein on gas vesicles surfaces, are clearly visible and can initiate immune responses.
additionally, the level of murine humoral responses observed likely would reflect effects from gas vesicle elicited cytokines and the natural slow but progressive release of antigen over time.
the complexity, functions and detailed interactions of cytokine and their network are well outside the scope of these studies; however in vitro effects of cell perturbation by gas vesicles and their siv derived inserts could establish a profile of cell responses relevant in the context of an antigen delivery system.
screenings for possible insert specific effects examined il- <dig>  il- <dig> and il- <dig> which have been implicated as protective during infection by intracellular pathogens  <cit> .
therefore studies quantified these three to obtain a profile of insert specific effects.
the assay results showed that despite the small size of the siv inserts, changes are evoked that differ quantitatively and temporally depending on the recombinant r-gv applied.
thus each had unique, signature effects when phagocytosed by cells and therefore would exert a different but characteristic impact when used as an immunogen.
these findings indicate that cytokine analyses that targeted examining the effect of individual peptide immunizing agents could be highly relevant as an additional parameter that aids in characterizing the epitope of those agents.
the in vitro time course examining r-gv uptake and fate used immunostaining with rabbit anti-gvpc and monkey anti-shiv antibodies to detect and assess a time course for intracellular breakdown of phagocytosed organelles.
the distribution of fluorescence labelling demonstrated the progressive loss over time of protein specific immunostaining.
as a larger protein species there was an inherently slower clearance of the gas vesicle protein, gvpc vs. the viral inserts which are small peptides.
in terms of in vivo host immune responses, slower degradation would extend the period of cell perturbation.
in turn this could impact cells and their processes - antibody production, specific antibody isotype generated, and the specific cytokine profiles elicited.
the complexity, functions and detailed interactions of cytokines and their network were outside the scope of these studies but the in vitro effects of cell perturbation by gas vesicles indicated screening for possible insert specific effects on il- <dig>  il- <dig> and il- <dig> would be a highly relevant parameter in defining the siv epitopes of interest here  <cit> .
these cytokines therefore were quantified to obtain a profile of insert specific effects.
the results showed r-gvtat, rev and nef <dig> each evoke changes that are quantitatively and temporally different.
therefore each likely would have unique, signature effects when phagocytosed by cells and thus would exert different but characteristic perturbations.
the impact of th1/th <dig> balance on immune responses to antigen perturbation is crucial: th <dig> responses supports inflammation and t and macrophage cell activation, while th <dig> actives primarily b cells, resulting in antigen specific antibody.
the cytokine quantifications were carried out as described using the in vitro cell culture system and there were notable temporal and quantitative differences.
clearly responses generated in vivo vs. in vitro can differ significantly as the former is a far more complex environment.
thus, in the absence of in vivo data there is no valid basis to speculate about the effect of this mode of antigen presentation in vivo.
however, the findings suggest that cytokine analyses to establish the effect of individual peptide immunizing agents delivered by r-gv could be generally useful as an additional parameter characterizing epitopes that are being selected for vaccine usage.
cell uptake of r-gvtat, rev or nef <dig> results in characteristic; insert specific changes in the three cytokines assessed.
the precise mechanisms of immune response regulation in vivo are highly complex.
however, for particulate antigens, such as those tested here, internalization and degradation by dendritic cells or macrophages would be a critical initial step in the process.
the in vitro findings indicate that the highly stable gas vesicle particle is readily and rapidly internalized by macrophages such as the j774a.
<dig> cell line used here.
the in vitro time course demonstrating the intracellular breakdown used rabbit anti-gvpc and monkey anti-shiv antibodies.
it demonstrated the progressive loss of protein specific immunostaining over time and as expected, the larger nef <dig> insert was degraded more slowly than the tat and rev components.
the later stages of this process also were examined by western blot.
as expected, these clearly showed the array of molecular weight species formed during intracellular breakdown.
it demonstrated the progression of changes and verified that at the  <dig> h time, breakdown remained incomplete and thus would continue to perturb the cell.
as a larger protein species the inherent slower clearance gvpc vs. the viral insert likely reflects primarily the larger size of the gvpc component.
however, in terms of host immune responses, gvpc's slower disappearance, an indicator of degradation, likely results in an extended period of cell perturbation.
in vivo perturbation can impact processes such as stimulation of antibody production, the specific antibody isotype that ultimately is formed, and the changing profile of cytokines produced.
finally, the results overall support the conclusion that although the siv inserts tested here differ in amino acid sequences, expressed peptide size and in vivo function during infection, both the western blot data, and the fluorescent microscopy studies show the inserts do not significantly alter the time course for r-gv degradation.
interestingly, intact rev and also tat proteins would normally track into the nucleus during in vivo infection by hiv  <cit> .
the immunostained  <dig> h micrographs, demonstrate that numerous cell nuclei specifically reacted with the anti-shiv antibody, some even exhibiting a green "glow".
thus data from the r-gvsiv treated monolayer cultures suggest that in this in vitro system, the rev peptides in particular, but also tat can enter the nuclear compartment of j774a cells.
potentially therefore, an inherent characteristic, amino acid sequence remains fully functional when expressed in gvpc-siv protein chimeras and effectively targets the nucleus.

 twenty eight years after the first cases were recognized, the hiv- pandemic continues to grow exponentially resulting in more than million cases of individuals living with hiv worldwide. constant virus replication in cd t lymphocytes initiates progressive immune defects and finally, after to years, results in acquired immunodeficiency syndrome and death. the course of the hiv infection has changed significantly with the development of new antiretroviral regimens that combine inhibitors of reverse transcription, virus protein cleavage, or even virus entry. they reduce viral burden and immune damage caused by hiv but cannot fully eradicate the virus. thus, lifelong therapy is expected to transform this otherwise lethal disease into a chronic, continuously treated infection by preventing the progression to aids. however, severe drug-related adverse effects and the development of drug resistance limit their efficacy, and the drugs have not been affordable for the vast majority of patients worldwide. because a therapeutic breakthrough that would soon eradicate hiv or limit side effects appears unlikely at present, additional therapeutic strategies continue to be relevant to the lasting prevention of aids onset. a better characterization of the initial host immune response to hiv- infection may help to define protective immunity to hiv- one such strategy might be to combine antiretroviral treatment with immune responses to hiv. some immune control of hiv is evidenced by the temporal association of virus reduction and the emergence of hiv-specific t cells, however in the absence of a pre-infection stimulus, anti-hiv neutralizing antibodies normally develop too late to play a key role during natural infections. findings have suggested that cellular immunity is involved in the initial control of virus replication in primary hiv- infection and indicate a role for ctl in protective immunity to hiv- in vivo. importantly, analyses of vaccination studies in nonhuman primate have indicated that single viral epitope-specific ctl responses may not be sufficient to block infection with pathogenic siv. in turn this suggests that the generation of broader responses that target multiple viral epitopes may be critical to the development of effective protection against aids. thus, a recent alternative approach has involved the use of multiple hiv antigens and the inclusion of both structural and regulatory antigens. an indication that this approach can be more effective has recently been verified in that in animals, protection from heterologous shiv challenge was observed only with immunization using an aggregated, multiantigen subunit protein vaccine that incorporated the structural protein env and the regulatory proteins tat and nef. when individually immunized using the same antigens, animals were not protected. additionally, studies demonstrated improved protection from aids and cd4+t-cell depletion in animals immunized with a combination of antigens. inherently, the microparticulate gas vesicle organelles produced by halobacterium sp. nrc- have numerous characteristics which would be desirable in an antigen presenting system and thus potentially can have importance for applications in vaccine development. the organelles are readily amenable to genetic engineering techniques that produce gas vesicles expressing on their surfaces the selected exogenous amino acid sequences, and the presence of surface borne peptide sequences has been assessed and verified in vivo. unlike most phage or animal virus-derived vaccine components, the gas vesicle structure does not contain nucleic acid. as a result, with the proteinaceous gas vesicle system the potential effects associated with co-introduction of dna sequences, inherent in viral or phage based vaccines, can be completely avoided. the gas vesicles are readily, rapidly and inexpensively isolated from the bacteria by lysis in low salt and the isolated nanoparticles are uniquely stable. large-scale production of gas vesicles is simple since these organelles float upon release from the halobacteria and thus are easily harvested. since they are non-toxic to animals and humans, gas vesicles should have utility for use as oral as well as parenteral delivery vehicles. as initially demonstrated, gas vesicles from a mutant halophilic archaea sd can be engineered to display various peptides by insertion of exogenous pathogen dna sequences into a site engineered into the gvpc gene. thus, expression system plasmids were generated in which sivsm gene segments were inserted into site "d" engineered within gvpc gene. the plasmid constructs were subsequently used to transform the vac- mutant, halobacterium nrc- strain sd and the recombinant gas vesicles harvested and isolated as described in detail previously. these nanoparticles were then pre-tested for immunogenicity, antigenicity and the activation of immunologic memory. in the present study we examined sera from different times post-booster and post re-immunization to assess the antibody isotype elicited in vivo by populations of gas vesicles each expressing a selected sivsm peptide sequence. using an in vitro macrophage cell culture system we then tested for possible effects of gas vesicle uptake on cytokines produced by cultured macrophages through quantification of three key examples: il- il- and il- finally, given the known, ultra stable nature of the gas vesicle nanoparticles we used in vitro culture in conjunction with immunofluorescence microscopy and western blot assessments to examine sivsm and gvpc molecular weight species as indicators of their degradation. thus, the changing intracellular distribution patterns of gvpc positive protein and the incorporated sivsm peptide, as well as the molecular weight species of degradation products were specifically examined.  immunoglobulins are glycoproteins present in serum and tissue fluids that recognize and bind their cognate antigen. they constitute the adaptive, humoral immune responses, are non invasively accessible for testing and their utility in vivo is in part a function of their specific isotype. as a first step in the present studies, we therefore evaluated our archived serum to assess antibodies elicited by recombinant gas vesicles displaying an sivsm peptide inserts as a function of immunization status and overall elapsed time post-immunization or booster. isotyping of immune sera based on results from the preliminary sera screenings, igg igg2a and igm were selected for evaluation utilizing the southernbiotech isotyping kit as described in the methods. the assay results are presented graphically in figure which plots the isotypes detected at the optimum antigen specific antibody dilution. this antigen specific approach allowed determining the predominant isotype elicited and present at different times post-immunizaton or booster. as the histograms in panels of figure show, with two exceptions, igg is the predominant isotype at and weeks post second booster. seventeen weeks later, the mice were re-immunized using aliquots of the same r-gv preparations and sera collected at days and again at weeks post re-immunization. the isotyping results for samples from these time points, shown in figure confirm that for all the mice tested, igg remains the predominant isotype including the assessments of sera drawn at weeks post re-immunization. for sera of all animals, at the optimum antigen specific dilution, igg remained the predominant isotype and its signal was greater than that of the day post re-immunization samples indicating the specific antibody responses continued to develop over time. determination of key cytokines elicited from j774a. macrophage cells in recent years the expression "cytokine network" has become commonly accepted in biomedical research and that network now includes several hundred members. using our in vitro monolayer system this study assessed the levels of three key cytokines as a function of r-gv uptake by macrophage cell monolayers. the studies had two aims: first to evaluate whether uptake of gas vesicle particles by these monolayer cells could influence the detectible levels of cytokines in the surrounding media and second, to determine whether there were differential cytokine levels as a correlate of the specific sivsm peptide expressed by the r-gvs applied to the individual monolayer cells. positive findings would add to a profile that characterizes peptide specific effects in vitro as well as to the potential utility of this nanoparticle delivery system. there are known interactive influences for each cytokine and macrophages normally produce il- il- and, il- among others. il- and il- have important roles in inducing th responses while il- can antagonize the generation of th subset of helper t cells and enhance proliferation of b cells. further, macrophages are widely distributed in mammalian tissues and fluids. therefore assessment utilized monolayers of j774a. murine macrophages and the protocols for in vitro r-gv application, followed by quantification of cytokines in the culture media as described in the methods section. to test whether there might be r-gv insert specific differences, cell monolayers were incubated for - h with wt-gv a control, or with the different r-gvtat, rev or nef at the times specified after gv removal and rinsing, aliquots of culture media were harvested and stored at -20°c until assessed for their content of the selected cytokines. cytokine quantifications used point to point curves generated by assay of the individual purified cytokines provided in the cytokine kits and the calculation methods recommended by the kit manufacturer. concentrations of the three different cytokines were assayed in media of cultured cells receiving no gas vesicles, media from duplicate cultures treated with one of the different sivsm insert containing r-gv populations, or with the insert negative wild type control from nrc- table presents these results which are snapshots in time of individual cytokine levels with the elevated levels shown in bold and shows there are notable differences in concentrations, and these vary as a function of the sivsm insert and also the time post application. thus for r-gvtat or revsamples, concentrations of il- and il- were maximal at h post application, then declined. for samples of r-gvnef the peak il- concentration was much higher and occurred at h; although lower in the h samples, it was still elevated. for il- maximal concentration again occurred early at h for r-gvtat treated cultures, then decreased to the level of the insert negative nrc- in the h sample. in contrast, for r-gvrev or nef media, increased pg/ml were detected only in the h samples with nef exhibiting the higher concentration. finally for il- samples from r-gvtat or rev treated cultures, the h samples displayed some elevation in concentration that somewhat exceeds the elevated levels detected in samples from the cultures exposed to the insert negative control, gas vesicles from nrc-. at later times, il- levels were additionally elevated by r-gvtat, with the higher levels occurring respectively at and h.. in r-gvrev treated samples, the h time displayed elevated il- but this decreased to ~half that concentration in the h and h post-addition samples. in contrast, at the different times tested, monolayers treated with r-gvnef displayed no significant increases in il- concentrations. in fact the levels were marginally lower than those from samples from insert negative nrc- cultures. therefore these assays indicate that unlike assays for r-gvtat and rev, r-gvnef elicited no significant insert specific increase in il- concentrations. † as detailed in the methods, macrophage monolayers received media only. ‡ control wells received the wild type gas vesicles nrc- although these data are "snapshots", nevertheless they indicate there are quantifiable, sivsm insert specific differences and time dependent effects on cytokines produced by j774a. cell following the application and internalization of r-gvtat, rev or nef thus in the context of an antigen delivery vehicle, there clearly is a detectible, insert specific impact on cytokine production by recipient monolayers. these results therefore add to an overall picture characterizing the immunogen as delivered via the r-gv system. they also suggest r-gvs could potentially be further designed for a co-delivery specifically aimed at simultaneously affecting cytokine levels. intracellular fate of gas vesicles at 50- nm, individual gas vesicle nanoparticles are below the limit of light microscope resolution but clusters of them, and of their breakdown products are detectible by immunostaining. gas vesicles are remarkably stable particles and thus may inherently result in a slow release of the incorporated peptide sequences. therefore r-gv, their fate and susceptibility to timely degradation constitute critical parameters in context of in vivo applications. immuno-microscopy assessment of r-gv intracellular fate studies directed at examining the post-phagocytic fate of the recombinant nanoparticles used specific antibody probes to detect the sivsm encoded peptides and the gvpc protein per se. this protein contains the insert and specifically identifies gas vesicle particles. using the protocols specified in the methods, the j774a. macrophage cell line monolayers were prepared and incubated with r-gv then fixed as described at the different, specified times and stored at -20°c for subsequent assessments. immunostaining with appropriate primary antibody allows immunofluorescent detection of r-gv delivered sivsm peptides -tat, rev or nef and separately, of the gas vesicle protein, gvpc. the initial distribution of r-gv material following internalization by monolayer cells is shown by the low magnification micrograph in figure it provides an overview of representative control and r-gvtat, rev and nef treated monolayers. these samples were harvested at h post r-gv treatment and subsequently immunostained with the anti-shiv monkey plasma which identifies the sivsm peptide. use of this plasma verifies that antibodies elicited during in vivo infection with live, native shiv recognizes the r-gvsivsm peptides synthesized by the halobacteria as if they were native siv protein. the monolayer in panel a of figure is a control to test for non specific antibody binding. it was not incubated with gas vesicle preparation and clearly the monolayer exhibits only a background fluorescence signal. in contrast, monolayers treated with r-gv show numerous cell associated fluorescence positive regions in tat and rev treated monolayers. for the larger nef insert, there also are cells that appear to "glow". this fluorescence labelling shows the more extensive binding of anti-shiv primary antibody to the larger nef peptide contained in r-gvnef particles. a second, critical characteristic is the susceptibility of the r-gv delivery vehicle to intracellular degradation that releases the siv inserts for processing by the cell. the fate of the sivsm insert was followed in immunostained samples harvested over a period of 12- h and representative × images of immounostain results are presented in figure as would be anticipated, these show that the sivsm specific staining appears to become more diffusely distributed in the cells both as a function of the elapsed time, and the specific insert. at h, shiv positive particles appear to be associated with the monolayer cells, internalized by them, often as a cluster or internalized to produce a somewhat more diffuse green fluorescence. by h the bright punctuate staining of rev and tat has diminished, becoming more diffuse, and these cells do not'glow'. however for nef the largest of the sivsm encoded insert tested, distinct regions of'glow' remain, likely because the degradation of this larger siv insert remains less complete. however by h, the immunostaining for nef also has become more generalized. interestingly, for the sample receiving r-gvrev, nuclear regions of the cells immunostain quite brightly and in the h rev sample, this apparent nuclear association is clearly evident. though not as distinct, a similar nuclear association occurs with the tat insert. in contrast, the r-gvnef treated sample does not exhibit this distribution. additionally, at the h time, the diffuse, lightly stained areas detected in the cytoplasm of the r-gvnef sample appear similar to the cytoplasmic distributions detectible in the h r-gvtat sample. this later appearance in the r-gvnef treated monolayer might be a function of slower breakdown of this larger expressed peptide. as expected, for each of the three different r-gv populations, the immunostaining of the incorporated sivsm derived inserts is consistent with ongoing r-gv breakdown of the individual clusters and concomitant disappearance of detectible, brightly fluorescent particle clusters. other companion monolayers were similarly rinsed, fixed, then immunostained using anti-gvpc specific antibody to detect the larger, gas vesicle specific "c" protein and figure presents a temporal series of representative images from 12- h post r-gv application. relative to the sivsm inserts, gvpc is a large protein and like gas vesicle proteins in general, is resistant to dissociation/degradation. immunostaining to identify gvpc initially gives rise to cells exhibiting an overall glow that becomes significantly diminished by the h time point and this would be consistent with a slow degradation of gvpc. however, positive but muted and somewhat diffuse punctuate immunostaining remains at the h time and can support suggesting that full degradation of gvpc remains incomplete at this time. in the context of a display/delivery particle, it is important that cells can both internalize the r-gvs, and successfully degrade them, allowing release of the pathogen specific insert they carry. the observed level and distribution of immuno-reactive gvpc material in monolayers at the h time is fully consistent with ongoing particle degradation. additionally, the decreasing brightness of the immunostaining supports suggesting the degradation time frame for and perturbation by gvpc will extend beyond the time required for the degradation of sivsm inserts. western blot assessments verify intracellular breakdown of sivsm and gvpc components of r-gv ultimately, the processing and removal of immunogens and their carriers is a significant factor for in vivo applications. therefore the fate of the gvpc and sivsm components detected by immuno fluorescence microscopy during the late stages post r-gv application were examined further. based on the patterns observed in these preparations, samples from the h and h were additionally assessed to verify protein breakdown. other j774a. monolayers were incubated with the gas vesicle preparations under the same conditions, then rinsed and harvested. these samples were assessed by preparing cell lysates and separating the protein species using 4-12% sds-page gradient gels as detailed in the methods. in acrylamide gels, pure intact gvpc protein behaves anomalously relative to its actual molecular weight, migrating as a species of ~ kda. following separation using sds-page, and transfer to pvdf membrane, gvpc peptides and the expressed sivsm insert were detected using the same anti-gvpc antibody stock and anti-shivsm monkey plasma utilized for the monolayers in immunostaining studies; companion halves of blot membranes were probed with the respective specific antibodies. figure presents typical western blots of samples from the h and h time points, probed respectively with anti-gvpc antibody or anti-shivsm plasma. gvpc is synthesized by the recombinant bacteria and an integral, surface displayed component of these gas vesicles. the observed immunostained bands of differing kda are a reflection of the molecular weights of gvpc breakdown products; other associated degradation resistant gas vesicle structural proteins also would be expected and if still integrated with gvpc, would affect its migration in gels. the presence of multiple protein bands identified for r-gv samples and for the shiv peptide is consistent with the immunostain microscopic observations which also suggested r-gv break down is a process that continues over a period of days. the membrane half probed with anti-gvpc antibody specifically identifies gvpc protein and peptides. it detected a number of bands at a variety of molecular weights, rather than the single ~ kda band that would characterize intact gvpc protein. the majority of bands in blots of the and h samples appear at < ~ kda with heavily stained bands detected at ~ kda and at somewhat at less than kda. the immunostain of these bands is notably stronger at the h time, indicating gvpc breakdown is continuing and gives rise to these species, but clearly is still incomplete. importantly, some bands may exhibit anomalous migration due to gas vesicle protein resistance to the standard protein dissociation protocols. slow disassembly and intracellular degradation would not be unexpected for gas vesicle proteins and likely perturbation of cells by partially degraded gvpc fragments would continue for an additional, but finite period of time. in contrast, the companion sivsm inserts of rev, tat and nef remain detectible but for the h sample, the peptides immunostain lightly with the anti-shiv antibody. notably the inserts are an integral part of gvpc and like other gv proteins, gvpc is resistant to degradation. multiple but light staining bands detected with the anti shiv antibody would therefore be expected. at h, the bands recognized by anti-shiv antibody are minimally in evidence. no immunoreactive high molecular weight bands are obvious and the molecular weight species of ~ kda or less now are only minimally detectible. clearly therefore, despite their integration into the original gvpc framework protein, the sivsm inserts are accessible to degradative processing. therefore these foreign proteins ultimately should be cleared by cells. discussion the studies presented above were designed to further assess practical aspects of the recombinant gas vesicle display/delivery system through in vitro and in vivo studies. as a self-adjuvanting, highly stable component, r-gv have significant potential as an alternative delivery system and thus the studies reported were designed to provide additional characterizations. the siv fragments tested were selected because they are key immunogens examined as relevant gene products in other published research those studies had shown toxoids of tat or tat and rev, or delivery of aggregated multiantigens, provided some disease attenuation whereas use of tat alone for example, provided only temporary infection containment. this was interpreted to mean a single antigen might not be adequate to provide full and/or long term protection. our recombinant system inherently supports the expression and delivery of various epitopes and could be readily adapted if important, new peptides were identified. thus combinations of r-gv particles could be easily created for use in vivo and this would provide exposure to multiple antigen fragments and thereby elicit a variety of peptide specific antibodies. different isotypes bind to cell surface expressed fc receptors in vivo and the resulting receptor derived cell stimulation then will initiate a cascade of effects that can impact the local milieu, and ultimately cell functions. utilizing the optimum antigen specific dilution of each antibody sample as determined by pre-titration using the specific tat, rev, and nef antigens, the present studies identified the isotype elicited by r-gv immunizations. these analyses showed igg was the predominant immunoglobulin species. although for the r and n groups, igm was predominant at days, at weeks, in the absence of any subsequent booster, igg was the dominant isotype in all samples. this can be relevant in vivo since antibody isotypes impact downstream events through antibody binding to cell displayed fc receptors. additionally, the continued presence of antigen specific mouse antibodies days after the final re-immunization, is potentially an important feature for an immunizing agent and likely to be relevant in terms of practical utility. the isotyping data also inherently demonstrated that the peptides encoded by the expressed small dna inserts, are immunogenic and as shown in figure relative to the expressed nef encoded insert appear to be degraded more slowly than their actual size would predict. this finding may reflect the central location of these encoded small peptide inserts, and amino acids respectively, within the gvpc protein. at that site they may be less readily accessible to the intracellular degradation processes. nevertheless they are immune visible and evoke specific antibody responses. gas vesicles are particulate moieties and when phagocytosed can impact various cell functions, including cytokine production. in turn, both individually and in combination, cytokine levels significantly affect the nature and level of immune responses and response polarization in terms of antibody, and cell mediated immunity. in vivo, cytokines impact immune related processes and certainly specific roles for different cytokines have been identified and extensively investigated. studies of hiv for example, have demonstrated cytokine correlations with protection from infection, prediction of disease progression and impact on immune response polarization. based on the potential for an insert specific effect, the present in vitro studies assessed the r-gv immunogens for their possible impact on selected cytokines, quantified by testing the media of monolayers treated with r-gvtat.rev and nef vs. wt-gv, or left untreated. although the siv specific peptide inserts constitute a very small segment in the recombinant gvpc protein, nevertheless in vitro studies showed there were detectible, insert specific differences in the cytokine patterns and the temporal profiles of their changing concentrations. given the known effects of cytokines il- il- and il- and data in the literature relating to tat, ref and nef the present findings show that despite the preponderance of gvpc specific protein, even very small segments of these pathogen proteins could play specific and significant roles in vivo by influencing the local milieu. although in vitro effects can not fully reflect in vivo process or responses, they nevertheless can indicate the potential to exert an effect. analogous consequences in vivo could alter the local milieu and thereby alter the th1/th responses equation. in turn, this might affect vaccine efficacy and certainly adds to a characterization of this immunization vehicle. thus similar examinations would be relevant in the broader context of vaccine development, and of characterizing pathogen impact on tissues or systems. known examples are inhibitory effects of il- on hiv- production in monocytes/macrophages resulting from il- stimulated inhibition of cytokine syntheses e.g. tumor necrosis factor alpha and il- in the context of hiv- this alteration can up-regulate pathogen expression in the infected cells. additionally, studies by others also have described il- inhibitory effects on cytokine synthesis, and on pathogen replication in monocytes. the inherent physical stability of the gas vesicle structure, along with its resistance to chemical dissociation or proteolytic cleavage and its particulate structure, likely play a role in the natural slow release of the incorporated epitope containing fusion proteins and could enhance stimulation of the immune system. ultimately however, the full degradation of the particulate r-gv immunogen is important. their long term presence potentially could elicit chronic, local inflammatory responses that can characterize degradation resistant particles. the in vitro immunostaining and the western blot findings presented here were aimed at assessing intracellular gv breakdown. the results indicate that degradation of these naturally stable immunogenic particles does occur and demonstrates the sizes of degradation products. in this context, it is noteworthy that even long term, oral ingestion of gas vesicles on a regular basis has provided no evidence that gas vesicles exhibit any detectible short or long term toxicity. thus per-oral usage of r-gv also could be an option. micrographs from the immunofluorescence staining allow the tracking of r-gv intracellular fate after the application to j774a. cells monolayers in vitro. these studies demonstrated that as expected, r-gv are phagocytosed. microscope focusing indicated r-gv associated with and/or were within j774a. macrophages at h post application and clearly internalized at later times. results from the subsequent immunostain monitoring of both the gas vesicle protein gvpc, and the siv specific inserts confirmed their slow, progressive disappearance over time. an intracellular accumulation/coalescence of individually internalized r-gv particles clearly takes place and is evident in the micrographs of h post application monolayers. additionally, samples from various times following removal and rinsing off of the applied r-gvtat, rev or nef were immunostained using either anti- shiv or anti-gvpc antibody and examined. these clearly show a decrease in detectible immunostained material during this time course. the results from this group of studies therefore provides critical information in terms of potential r-gv utility as a delivery vehicle since it indicates that in vivo there should be an ongoing but distinctly finite host cell perturbation following cellular uptake. this likely would impact immune stimulation, potentially by processes including the stimulation of cytokine production detected and quantified above. importantly the present studies tracking degradation indicated that over time in vitro, the recombinant particles did undergo degradation in the individual cells. this was evidenced by the immunomicroscopy data for samples and was verified in more detail by the western blot assessments which tested lysates of r-gv treated monolayer at and h post r-gv applications. since recombinant inserts are expressed in gas vesicle protein'c', studies assessed breakdown of this protein species and also of the specific expressed sivsm inserts. in sds-page gels isolated, intact gvpc would appear as a single, ~ kda protein species. the western blot analysis using anti-gvpc showed clearly that wt-gv and the r-gvtat, rev and nef recombinants all exhibited multiple molecular weight species at both and the h times after application to monolayer cells. this is the predicted result when proteins that become internalized are broken down and is consistent with the immunostaining detected in figures and it also would be expected that the band patterns in sds-page gels, and the density of western blot immunostained bands would change as a function of time and this is the case. thus, by h, there are crisp gvpc positive bands of lower molecular weight species, as well as higher molecular weight species which are now more lightly stained by the anti-gvpc antibody. all of these bands are reactive with the anti-gvpc antibody, and since in sds-page gels, gvpc that is un-degraded behaves as a kda moiety; these clearly are degradation products from the gvpc protein. they exhibit discrete, decreasing molecular weights consistent with degradation that is ongoing over time. similarly the companion western blots probed with anti-shiv specific antibody demonstrated breakdown of the sivsm derived inserts and showed the disappearance of bands over time. in addition to the detailed findings, the results overall support the conclusion that intracellular r-gv are susceptible to degradation and therefore can be cleared from cells that may phagocytose them. furthermore, unlike the effects on cytokine concentrations, inserts in r-gvtat, rev or nef do not appear to significantly alter the r-gv time course of degradation in an insert dependant way.  the studies presented here provide an initial examination of recombinant gas vesicles in terms of predominant antibody isotype elicited in vivo, particle impact on selected cytokines elicited following uptake by monolayer cells in vitro and r-gv internalization and intracellular degradation during in vitro culture. the murine immunizations showed that despite the relatively small size of the expressed recombinant insert, like other haptens sivsm derived inserts incorporated into the gvpc "carrier" protein on gas vesicles surfaces, are clearly visible and can initiate immune responses. additionally, the level of murine humoral responses observed likely would reflect effects from gas vesicle elicited cytokines and the natural slow but progressive release of antigen over time. the complexity, functions and detailed interactions of cytokine and their network are well outside the scope of these studies; however in vitro effects of cell perturbation by gas vesicles and their siv derived inserts could establish a profile of cell responses relevant in the context of an antigen delivery system. screenings for possible insert specific effects examined il- il- and il- which have been implicated as protective during infection by intracellular pathogens. therefore studies quantified these three to obtain a profile of insert specific effects. the assay results showed that despite the small size of the siv inserts, changes are evoked that differ quantitatively and temporally depending on the recombinant r-gv applied. thus each had unique, signature effects when phagocytosed by cells and therefore would exert a different but characteristic impact when used as an immunogen. these findings indicate that cytokine analyses that targeted examining the effect of individual peptide immunizing agents could be highly relevant as an additional parameter that aids in characterizing the epitope of those agents. the in vitro time course examining r-gv uptake and fate used immunostaining with rabbit anti-gvpc and monkey anti-shiv antibodies to detect and assess a time course for intracellular breakdown of phagocytosed organelles. the distribution of fluorescence labelling demonstrated the progressive loss over time of protein specific immunostaining. as a larger protein species there was an inherently slower clearance of the gas vesicle protein, gvpc vs. the viral inserts which are small peptides. in terms of in vivo host immune responses, slower degradation would extend the period of cell perturbation. in turn this could impact cells and their processes - antibody production, specific antibody isotype generated, and the specific cytokine profiles elicited. the complexity, functions and detailed interactions of cytokines and their network were outside the scope of these studies but the in vitro effects of cell perturbation by gas vesicles indicated screening for possible insert specific effects on il- il- and il- would be a highly relevant parameter in defining the siv epitopes of interest here. these cytokines therefore were quantified to obtain a profile of insert specific effects. the results showed r-gvtat, rev and nef each evoke changes that are quantitatively and temporally different. therefore each likely would have unique, signature effects when phagocytosed by cells and thus would exert different but characteristic perturbations. the impact of th1/th balance on immune responses to antigen perturbation is crucial: th responses supports inflammation and t and macrophage cell activation, while th actives primarily b cells, resulting in antigen specific antibody. the cytokine quantifications were carried out as described using the in vitro cell culture system and there were notable temporal and quantitative differences. clearly responses generated in vivo vs. in vitro can differ significantly as the former is a far more complex environment. thus, in the absence of in vivo data there is no valid basis to speculate about the effect of this mode of antigen presentation in vivo. however, the findings suggest that cytokine analyses to establish the effect of individual peptide immunizing agents delivered by r-gv could be generally useful as an additional parameter characterizing epitopes that are being selected for vaccine usage. cell uptake of r-gvtat, rev or nef results in characteristic; insert specific changes in the three cytokines assessed. the precise mechanisms of immune response regulation in vivo are highly complex. however, for particulate antigens, such as those tested here, internalization and degradation by dendritic cells or macrophages would be a critical initial step in the process. the in vitro findings indicate that the highly stable gas vesicle particle is readily and rapidly internalized by macrophages such as the j774a. cell line used here. the in vitro time course demonstrating the intracellular breakdown used rabbit anti-gvpc and monkey anti-shiv antibodies. it demonstrated the progressive loss of protein specific immunostaining over time and as expected, the larger nef insert was degraded more slowly than the tat and rev components. the later stages of this process also were examined by western blot. as expected, these clearly showed the array of molecular weight species formed during intracellular breakdown. it demonstrated the progression of changes and verified that at the h time, breakdown remained incomplete and thus would continue to perturb the cell. as a larger protein species the inherent slower clearance gvpc vs. the viral insert likely reflects primarily the larger size of the gvpc component. however, in terms of host immune responses, gvpc's slower disappearance, an indicator of degradation, likely results in an extended period of cell perturbation. in vivo perturbation can impact processes such as stimulation of antibody production, the specific antibody isotype that ultimately is formed, and the changing profile of cytokines produced. finally, the results overall support the conclusion that although the siv inserts tested here differ in amino acid sequences, expressed peptide size and in vivo function during infection, both the western blot data, and the fluorescent microscopy studies show the inserts do not significantly alter the time course for r-gv degradation. interestingly, intact rev and also tat proteins would normally track into the nucleus during in vivo infection by hiv. the immunostained h micrographs, demonstrate that numerous cell nuclei specifically reacted with the anti-shiv antibody, some even exhibiting a green "glow". thus data from the r-gvsiv treated monolayer cultures suggest that in this in vitro system, the rev peptides in particular, but also tat can enter the nuclear compartment of j774a cells. potentially therefore, an inherent characteristic, amino acid sequence remains fully functional when expressed in gvpc-siv protein chimeras and effectively targets the nucleus. 
strain sd expressing cassettes with different sivsm inserts, have potential utility as an effective antigen display system for immunogen testing in vivo and for initial epitope assessments in vitro. humoral responses to r-gvtat, rev or nef elicited in vivo, associated changes in selected cell cytokine production following r-gv internalization, and the capacity of j774a. the in vivo studies involving r-gv immunizations and in vitro studies of r-gv uptake by j774a. recombinant gas vesicles from halobacterium sp. these features support the cost effective generation of vaccine components, and provide a simple, self-adjuvanting system for assessing immune visibility of and specific responses to individual pathogen peptides. macrophages demonstrated: tests for antibody isotypes in immunized mice sera showed activation and re-stimulation of memory b cells, during long term immune response to the epitopes, primarily the igg isotype was produced, in vitro, macrophage degradation of r-gv containing different sivsm inserts occurred over a period of days resulting in an inherent slow breakdown and degradation of the sivsm peptide inserts, vesicle specific gvpc, a larger protein, degraded more slowly than the recombinant peptide inserts and in vitro uptake and degradation of the r-gv populations tested was associated with sivsm insert specific patterns for cytokines il- il- and il- they can clearly: display various exogenous peptides, be intracellularly degraded in vitro over a period of days, affect cell cytokine levels, and retain their self-adjuvanting capacity irrespective of the specific peptide expressed within the gvpc protein.
 recombinant gas vesicles from halobacterium sp. strain sd expressing cassettes with different sivsm inserts, have potential utility as an effective antigen display system for immunogen testing in vivo and for initial epitope assessments in vitro. previous mouse model studies demonstrated immunization with r-gv expressing selected exogenous sequences elicited a prolonged immune response. here we tested segments from three sivsm genes each surface displayed by r-gv. as with hiv, for sivsm the proteins encoded by tat, rev and nef respectively serve critical and diverse functions: effects on efficient viral rna polymerase ii transcription, regulation of viral gene expression and effects on specific signaling functions through the assembly of multiprotein complexes. humoral responses to r-gvtat, rev or nef elicited in vivo, associated changes in selected cell cytokine production following r-gv internalization, and the capacity of j774a. macrophage cells to degrade these internalized display/delivery particles in vitro were examined.  the in vivo studies involving r-gv immunizations and in vitro studies of r-gv uptake by j774a. macrophages demonstrated: tests for antibody isotypes in immunized mice sera showed activation and re-stimulation of memory b cells, during long term immune response to the epitopes, primarily the igg isotype was produced, in vitro, macrophage degradation of r-gv containing different sivsm inserts occurred over a period of days resulting in an inherent slow breakdown and degradation of the sivsm peptide inserts, vesicle specific gvpc, a larger protein, degraded more slowly than the recombinant peptide inserts and in vitro uptake and degradation of the r-gv populations tested was associated with sivsm insert specific patterns for cytokines il- il- and il-  together these findings provide new information underscoring r-gv potential. they can clearly: display various exogenous peptides, be intracellularly degraded in vitro over a period of days, affect cell cytokine levels, and retain their self-adjuvanting capacity irrespective of the specific peptide expressed within the gvpc protein. these features support the cost effective generation of vaccine components, and provide a simple, self-adjuvanting system for assessing immune visibility of and specific responses to individual pathogen peptides. 