Fred R. Frankel

Studies on the nature of replicating DNA in T4-infected Escherichia coli†

The replicating DNA of T4-infected Escherichia coli has been isolated as a rapidly sedimenting structure of about 200 s at 15 minutes after infection. This sedimentation, rate is approached from smaller values at earlier times after infection. Several properties suggest that it differs from ordinary molecules of phage DNA or nonspecific aggregates of phage DNA. The very fragile structure breaks into products that sediment like phage DNA but are less homogeneous and appear to be more sensitive to further breakage than phage DNA. Treatment with nucleases that act on denatured DNA results in the simultaneous formation of intermediate rapidly sedimenting material and heterogeneous fragments ranging from 30 s to 100 s. An early hydrolysis product sediments at 73 s. The latter resembles a DNA fraction recovered from chloramphenicol-treated phage infected cells ( Frankel, 1963 ). The buoyant density in CsCl of the isolated replicating DNA is almost identical with that of phage DNA. After recovery from the same density band, the two DNAs retain, in part, their difference in sedimentation rate. The replicating DNA is more sensitive to thermal denaturation than phage DNA, but after complete denaturation the densities of the two are identical. Preliminary experiments suggest that the polynucleotide strands of replicating DNA are not longer than those of phage DNA. It is concluded that T4 phage DNA in the replicating state has a structure that is different from mature phage DNA. The former may be longer and could contain all of the newly synthesized DNA in a single, bizarre structure. This structure would possess regions in which the usual base pairing of native DNA is interrupted. Other structures, however, are not excluded.

Tubulin and actin in paired nonneoplastic and spontaneously transformed neoplastic cell lines in vitro: Fluorescent antibody studies

Pairs of nonneoplastic and spontaneously transformed neoplastic cells were derived from rat, mouse and hamster embryos. The neoplastic cells of each pair had poorly spread cellular morphology, grew in agarose in vitro and produced invasive sarcomas in vivo; the nonneoplastic cells exhibited none of these properties. The distribution of microtubules and microfilament bundles (stress fibers or actin cables) was examined in five such paired lines and in 3T3 and SV40-transformed 3T3 cells by indirect immunofluorescent microscopy of fixed cells treated with rabbit antibody prepared against bovine brain tubulin or guinea pig smooth muscle actin, respectively. Actin cables in all the neoplastic cells appeared thinner and more sparse than in the paired nonneoplastic cells. These differences were also observed in living cells with polarization microscopy. In contrast, microtubules appeared similar in neoplastic and nonneoplastic cells, both in areas of thin peripheral lamellar cytoplasm which allowed a clear visualization of fine, curving microtubules and in regions of thick, central endoplasm which obsecured individual microtubules. In fact, the main morphological difference between neoplastic and nonneoplastic cells was the relative amount of lamellar cytoplasm or endoplasm, rather than the appearance of microtubles in either region. Thus the distinctive growth properties and retracted cellular morphology of neoplastic cells in this study did not correlate with decreased or disorganized microtubules, but with thin and sparse actin cables.

Induction of Human Immunodeficiency Virus (HIV)-Specific CD8 T-Cell Responses by Listeria monocytogenes and a Hyperattenuated Listeria Strain Engineered To Express HIV Antigens

ABSTRACT Induction of cell-mediated immunity may be essential for an effective AIDS vaccine. Listeria monocytogenes is an attractive bacterial vector to elicit T-cell immunity to human immunodeficiency virus (HIV) because it specifically infects monocytes, key antigen-presenting cells, and because natural infection originates at the mucosa. Immunization with recombinant L. monocytogenes has been shown to protect mice from lymphocytic choriomeningitis virus, influenza virus, and tumor inoculation.L. monocytogenes expressing HIV gag elicits sustained high levels of Gag-specific cytotoxic T lymphocytes (CTLs) in mice. We have examined the ability of Listeria to infect human monocytes and present HIV antigens to CD8 T lymphocytes of HIV-infected donors to induce a secondary T-cell immune response. Using this in vitro vaccination protocol, we show that L. monocytogenes expressing the HIV-1 gag gene efficiently provides a strong stimulus for Gag-specific CTLs in HIV-infected donor peripheral blood mononuclear cells.Listeria expressing Nef also elicits a secondary in vitro anti-Nef CTL response. Since L. monocytogenes is a pathogen, before it can be seriously considered as a human vaccine vector, safety concerns must be addressed. We therefore have produced a highly attenuated strain of L. monocytogenes that requiresd-alanine for viability. The recombinant bacteria are attenuated at least 105-fold. We show that when these hyperattenuated bacteria are engineered to express HIV-1 Gag, they are at least as efficient at stimulating Gag-specific human CTLs in vitro as wild-type recombinants. These results suggest that attenuatedListeria is an attractive candidate vaccine vector to induce T-cell immunity to HIV in humans.

Novel Vaccination Protocol with Two Live Mucosal Vectors Elicits Strong Cell-Mediated Immunity in the Vagina and Protects against Vaginal Virus Challenge

Most HIV infections result from heterosexual transmission to women. Because cellular immunity plays a key role in the control of the infection, we sought to strengthen cellular immune responses in vaginal tissue. We explored a novel prime-boost protocol that used two live mucosal agents that trigger different pathways of innate immunity and induce strong cellular immunity. Adenovirus serotype 5 (Ad5) has frequently been used as a boost for DNA vaccines. In this study we used attenuated, recombinant L. monocytogenes-gag (rLm-gag) to prime mice by various mucosal routes—oral, intrarectal, and intravaginally (ivag)—followed by a systemic or mucosal boost with replication-defective rAd5-gag. Mice primed with a single administration of rLm-gag by any route and then boosted with rAd5-gag intramuscularly exhibited abundant Gag-specific CD8 T cells in spleen and vaginal lamina propria. Conversely, when boosted with rAd5-gag ivag, the immune response was reoriented toward the vagina with strikingly higher CD8 T cell responses in that tissue, particularly after ivag immunization by both vectors (ivag/ivag). Five weeks to 5 mo later, ivag/ivag-immunized mice continued to show high levels of effector memory CD8 T cells in vagina, while the pool of memory T cells in spleen assumed a progressively more central memory T cell phenotype. The memory mice showed high in vivo CTL activity in vagina, a strong recall response, and robust protection after ivag vaccinia-gag challenge, suggesting that this prime-boost strategy can induce strong cellular immunity, especially in vaginal tissues, and might be able to block the heterosexual transmission of HIV-1 at the vaginal mucosa.

The role of gene 49 in DNA replication and head morphogenesis in bacteriophage T4

Abstract Mutations in T4 phage genes 33 and 55, genes necessary for the synthesis of late phage-induced proteins, cause the replicating phage DNA to appear in lysates in a form that sediments much more rapidly than the normal 200 s form observed during wild-type infection. We now find that an amber mutation in gene 49 produces this same effect. A temperature-sensitive mutation in gene 49 produces an intermediate DNA form, both at permissive and restrictive temperatubes, indicating that the unusual form of the DNA does not preclude its being converted to phage particles. Mutation in no other late gene tested produces this phenotype. However, we find that inhibition of late protein synthesis with chloramphenicol can partially simulate the effect of gene 33, 55 and 49 mutations. Mutations in gene 49 also prevent the formation of mature 63 s DNA. When the rapidly sedimenting form of replicating phage DNA is isolated and tested as substrate, extracts prepared after T4 infection convert it to slower sedimenting structures. The reaction produces 200 s DNA and apparently terminates with the formation of molecules that sediment between 45 and 65 s. Early-mutant extracts and gene 33-defective and 55-defective extracts do not contain this activity. All late-mutant extracts tested do contain the activity, except those prepared after infection with either of the gene 49 mutants. Suppression of the amber mutation in gene 49 results in a tenfold increase in activity, but the suppressed extracts still possess only 0.1 the wild-type activity. Extracts prepared after ts49 infection also show about 0.1 the wild-type activity, regardless of the temperature of infection or assay. Mixing experiments show that gene 49-defective extracts do not contain an inhibitor of the wild-type activity. Mixed infection with various ratios of 49- to 49+ phage produces extracts containing activity approximately proportional to the number of 49+ gene copies. Lysates from certain of these mixed infections contain both forms of intracellular DNA and therefore complementation is not as complete as expected of a catalytic function. Gene 49 appears to control a product which, when functional, produces 200 s DNA in lysates of infected cells, but when absent results in the appearance of the more rapidly sedimenting DNA form. This gene product may be an enzyme that functions to alter the structure of the intracellular DNA so that it can be cleaved or encapsulated to form phage particles. Since the activity that we detect in extracts is sensitive to anti-T4 antiserum but not to pre-immune serum, it may be a component of the phage particle itself.

Systemic Immunity and Mucosal Immunity Are Induced against Human Immunodeficiency Virus Gag Protein in Mice by a New Hyperattenuated Strain of Listeria monocytogenes

ABSTRACT Vaccines designed to control chronic infections by intracellular agents such as human immunodeficiency virus type 1 (HIV-1) require the induction of cell-mediated immune responses to rid the host of pathogen-infected cells. Listeria monocytogenes has characteristics that make it an attractive vaccine vector for use against such infections. Here we show that parenteral immunization with a new highly attenuated strain of this organism provided complete protection against systemic and mucosal challenges with a recombinant vaccinia virus expressing HIV-1 gag. Immunization also generated a strong, long-term memory cytotoxic-T-lymphocyte (CTL) response in spleen, mesenteric lymph nodes, and Peyers patches directed against the gag protein. Oral immunization with this attenuated strain also produced complete, long-lasting protection against the recombinant virus but only against mucosal virus challenge. Curiously, oral immunization was associated with a transient CTL response in the three lymphoid tissues examined.

The absence of mature phage DNA molecules from the replicating pool of T-even-infected Escherichia coli.

The replicating DNA pool contained in Escherichia coli infected with T-even bacteriophage was examined by chromatography of phenol-extracted lysates on columns of methylated albumin—kieselguhr and by zone sedimentation of unextracted lysates. The pool apparently contains few DNA molecules having the properties of DNA extracted from mature phage particles. Part of the replicating DNA resisted phenol extraction and part of the DNA that could be extracted was found to bind irreversibly to the chromatographic columns. If extraction was eliminated and the lysates examined by sedimentation, the bulk of the newly synthesized DNA sedimented considerably faster than mature phage DNA. These properties could be those of a large, fragile structure that contains regions of single-strandedness. In contrast with the newly synthesized DNA, the intrabacterial parental DNA immediately after infection could be identified as normal phage DNA molecules. These, however, seem to become modified in some way, since their recovery rapidly decreased with time after infection. A small part of the parental DNA became fragmented in the infected cell. A much smaller fraction of newly synthesized DNA was present as fragments. The difference between lysates produced by treating cells with detergent and treating cells first with lysozyme and then detergent is discussed, and some properties of the cells treated only with detergent are described.

Conditional Lethality Yields a New Vaccine Strain of Listeria monocytogenes for the Induction of Cell-Mediated Immunity

ABSTRACT Listeria monocytogenes is a gram-positive intracellular pathogen that can enter phagocytic and nonphagocytic cells and colonize their cytosols. Taking advantage of this property to generate an intracellular vaccine delivery vector, we previously described a mutant strain of L. monocytogenes, Δdal Δdat, which is unable to synthesize cell wall by virtue of deletions in two genes (dal and dat) required for d-alanine synthesis. This highly attenuated strain induced long-lived protective systemic and mucosal immune responses in mice when administered in the transient presence of d-alanine. We have now increased the usefulness of this organism as a vaccine vector by use of an inducible complementation system that obviates the need for exogenous d-alanine administration. The strain expresses a copy of the Bacillus subtilis racemase gene under the control of a tightly regulated isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter present on a multicopy plasmid. This bacterium demonstrates strict dose-dependent growth in the presence of IPTG. After removal of inducer, bacterial growth ceased within two replication cycles. Following infection of mice in the absence of IPTG or d-alanine, the bacterium survived in vivo for less than 3 days. Nevertheless, a single immunization elicited a state of long-lasting protective immunity against wild-type L. monocytogenes and induced a subset of effector listeriolysin O-specific CD11a+ CD8+ T cells in spleen and other tissues that was strongly enhanced after secondary immunization. This improved L. monocytogenes vector system may have potential use as a live vaccine against human immunodeficiency virus, other infectious diseases, and cancer.

Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges

We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus > 90%; these RM also had strong SIV Gag-specific proliferation of CD8⁺ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4⁺ T cells; the latter have been implicated as preferential virus targets in vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection.

Identification of the T4rIIB gene product as a membrane protein

Synthesis of membrane proteins was found to occur after T4 infection of Escherichia coli. Many of the T4-induced proteins were clearly absent from membranes isolated from uninfected cells. One of these proteins was shown to be genetically controlled by the T4 genome and identified as the primary product of the rIIB cistron. This protein was absent from membranes isolated from rIIB-infected cells, was present in membranes from rIIA-infected cells, was synthesized with delayed-early kinetics, and had a molecular weight that was close to that predicted for the rIIB product. In addition, certain rIIB deletion and frameshift mutants were found to cause the synthesis of membrane-bound peptides the molecular weights of which corresponded to the portion of the rIIB cistron expected to be translated in those mutants. Synthesis of the fragment produced by deletion mutant T4r187 showed delayed-early kinetics, was unaffected by deletion of the rIIA cistron, but was eliminated by additional alterations of the rIIB cistron. Tryptic peptide analysis of the membrane protein found after T4+ infection and of the polypeptide found after T4r187 infection demonstrated the common origin of these two proteins. An extra peptide derived from the wild-type protein probably corresponded to the portion of the rIIB cistron that was deleted in r187. An extra tryptic peptide derived from the r187 fragment may correspond to a portion of the adjoining cistron co-translated with the rIIB fragment. This analysis did not permit identification of the rIIA product, although there was an indication that it too may be a membrane-bound protein.