Canine rabies DNA vaccination: a single-dose intradermal injection into ear pinnae elicits elevated and persistent levels of neutralizing antibody

Rabid dog exposures cause >99% of human rabies deaths world-wide. In developing countries, where dogs are the viral reservoir, the 30-50% vaccination coverage of dog populations is insufficient to break the disease transmission cycle. In addition, many vaccines currently used in developing countries fail to maintain detectable levels of neutralizing antibody. The poor vaccination coverage with inadequate vaccines, in addition to the difficulty in locating dogs for booster vaccinations, suggest that an inexpensive vaccine that elicits long-term immunity after a single-dose vaccination could improve control of canine rabies in developing countries. One solution could be a DNA vaccine. This study was designed to evaluate in dogs the ability of different methods of a single-dose DNA vaccination to elicit enhanced levels of neutralizing antibody. Intradermal (i.d.) vaccination into ear pinnae elicited elevated and long-lasting levels of neutralizing antibody. Minimal or undetectable levels of neutralizing antibody were detected after vaccination into quadriceps muscle, gene gun vaccination into ear pinnae or i.d. vaccination into the neck. Intramuscular (i.m.) or gene gun vaccinations did not "immunologically prime" a majority of dogs vaccinated by these routes. The passive transfer of sera from dogs that had been vaccinated i.d. in ear pinnae protected mice against rabies virus challenge. A single-dose i.d. rabies DNA vaccination into ear pinnae could aid in the control of canine rabies in developing countries.     

Priming of piglets against enterotoxigenic E. coli F4 fimbriae by immunisation with FAEG DNA

Early vaccination is necessary to protect pigs against postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). However, at present no commercial vaccine allows successful vaccination. This is partly due to the presence of maternally derived antibodies. Since DNA vaccines are suggested to be superior to protein vaccines in young animals with maternal antibodies, we determined whether the fimbrial adhesin (FaeG) of F4ac(+) ETEC could be used as a plasmid DNA vaccine to prime piglets in a heterologous prime-boost approach. Hereto, pcDNA1/faeG19 was constructed and expression of rFaeG in Cos-7 cells was demonstrated. Thereafter, pigs were immunised (days 0, 21 and 42) intramuscularly by injection or intradermally by gene gun and humoral and cellular immune responses were analysed. Even though responses were low, results demonstrated that intramuscular injection was superior to gene gun delivery for priming the humoral immune response since higher antibody titres were raised, whereas gene gun delivery better induced a cellular response, evaluated by a lymphocyte proliferation assay. Effective priming of the humoral immune response was evidenced by high IgG titres 1 week after a protein boost with purified F4. The low responses to the pcDNA1/faeG19 DNA vaccination suggest that delivery of the DNA and/or the expression of the faeG gene should be improved.     

One-time gene gun or intramuscular rabies DNA vaccination of non-human primates: comparison of neutralizing antibody responses and protection against rabies virus 1 year after vaccination.

We have previously shown that Macaca fascicularis (Cynomologus) monkeys receiving a primary and either one or two booster rabies DNA vaccinations are protected against rabies virus. In this study, we determined whether monkeys that had been vaccinated only once via gene gun or intramuscularly (i.m.) with different concentrations of DNA would be protected against rabies virus challenge. Neutralizing antibody responses were assayed for 1 year before the monkeys were challenged. Neutralizing antibody was detected at least 50 days earlier in gene gun vaccinated as compared to i.m. vaccinated animals. Prior to viral challenge, all (6/6, 100%) gene gun vaccinated animals, but only 3/6 (50%) i.m. vaccinated animals seroconverted. In general, antibody titers of the gene gun vaccinated animals were higher than the titers of the i.m. vaccinated animals. There was no correlation between the concentration of DNA used for vaccination, the neutralizing antibody responses elicited and protection against viral challenge. Seven days after viral challenge, a rapid and strong anamnestic antibody response was elicited in 100% of the gene gun vaccinated monkeys and in four i.m. vaccinated monkeys. Neutralizing antibody remained undetectable in two i.m. vaccinated monkeys. Overall, 60% (3/5) of the gene gun vaccinated animals and 87% (5/6) of the i.m. vaccinated monkeys survived viral challenge. This study is the first, to our knowledge, to show long-term protection of non-human primates against a human viral pathogen using a DNA vaccination protocol that did not include a booster immunization.     

Efficacy of DNA-hsp65 vaccination for tuberculosis varies with method of DNA introduction in vivo

A DNA vaccine codifying the mycobacterial hsp65 can prevent infection with Mycobacterium tuberculosis in a prophylactic setting and also therapeutically reduce the number of bacteria in infected mice. The protective mechanism is thought to be related to Th1-mediated events that result in bacterial killing. To determine the best method of hsp65 introduction for vaccination efficacy against tuberculosis (TB), we evaluated the immunogenicity and protection of DNA-hsp65 administered by gene gun bombardment or intramuscular (i.m.) injection of naked DNA. Immunization by gene gun induced immune response with plasmid doses 100-fold lower than those required for intramuscular immunization. However, in contrast to intramuscular immunization, which was protective in these studies, gene gun immunization did not protect BALB/c mice against challenge infection.     

Rabies DNA vaccination of non-human primates: post-exposure studies using gene gun methodology that accelerates induction of neutralizing antibody and enhances neutralizing antibody titers

Pre-exposure DNA vaccination protects non-human primates against rabies virus. Post-exposure protection of monkeys against rabies virus by DNA vaccination has not been attempted. Presumably, post-exposure experiments have not been undertaken because neutralizing antibody is usually slow to be induced after DNA vaccination. In this study, we initially attempted to accelerate the induction of neutralizing antibody by varying the route and site of DNA vaccination and booster frequency. Gene gun (GG) vaccinations above axillary and inguinal lymph nodes or in ear pinnae generated higher levels of neutralizing antibody than intradermal (ID) needle vaccinations in the pinnae. Concurrent GG booster vaccinations above axillary and inguinal lymph nodes and in ear pinnae, 3 days after primary vaccination, accelerated detectable neutralizing antibody. GG booster vaccinations also resulted in higher neutralizing antibody levels and increased the durability of this response. Post-exposure vaccination with DNA or the human diploid cell vaccine (HDCV), in combination with an one-time treatment with human rabies immune globulin (HRIG), protected 50 and 75% of the monkeys, respectively, as compared to 75% mortality of the controls. These data will be useful for the refinement, development, and implementation of future pre- and post-exposure rabies DNA vaccination studies.     

Immunisation against PCV2 structural protein by DNA vaccination of mice

Porcine circovirus type 2 (PCV2) is the causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome (PMWS). The disease affects primarily 5-12-weeks-old pigs which might suggest that infection with PCV2 occurs when the level of maternal antibodies have declined to sub-protective levels around weaning at 3-5-weeks of age. If immunoprophylaxis is to be effective, an immunisation method capable of breaking through maternal immunity must be employed. In this study, we have developed and investigated the potential of a DNA vaccination approach to be one such method. The gene encoding the capsid protein of PCV2 was cloned in a DNA vaccination plasmid and expression of capsid protein was demonstrated in vitro. Mice were gene gun vaccinated three timesand all mice responded serologically by raising antibodies against PCV2. The results suggest, that DNA based vaccination might offer opportunities for vaccination of piglets against PCV2.     

DNA vaccination of mice against rabies virus: effects of the route of vaccination and the adjuvant monophosphoryl lipid A (MPL)

Adjuvants are known to strongly enhance immune responses generated by traditional vaccines, but less is known about the effects of adjuvants on vaccination with DNA. In this study, we investigated the use of the immunostimulant monophosphoryl lipid A (MPL(R)) as an adjuvant, and analyzed three routes of DNA vaccination to determine if this adjuvant could enhance anti-rabies virus neutralizing antibody responses. Compared with antibody titers elicited with DNA only, antibody titers were enhanced after initial intradermal (i.d.) and gene gun immunizations with the combination of DNA and MPL(R). Antibody was not detected after primary intramuscular (i.m.) immunization unless MPL(R) was included with the DNA. Surprisingly, antibody titers of MPL(R)-treated mice decreased after i.d. or i.m. booster vaccinations, but increased after gene gun booster vaccinations. In contrast to these varied responses, booster immunizations without MPL(R) via the three different routes consistently increased antibody titers. All mice with detectable levels of neutralizing antibody at the time of challenge survived virus infection. There was no difference in the survival rate between groups of mice that received similar vaccinations with MPL(R)/DNA or DNA only. The data suggest that MPL(R) can enhance the neutralizing antibody response when used with the initial injection of DNA. Suppression of neutralizing antibody responses after i.d. or i.m. booster vaccinations that included MPL(R) suggests that the number of vaccinations, and the route of vaccination, should be carefully considered when MPL(R) is used with DNA vaccines.     

Protection of ferrets against influenza challenge with a DNA vaccine to the haemagglutinin

Immunization of ferrets with a plasmid DNA expressing influenza virus haemagglutinin (pCMV/H1 DNA) provided complete protection from challenge with the homologous A/PR/8/34 (HINI) influenza virus. Delivery of DNA-coated gold beads by gene gun to the epidermis was much more efficient than intramuscular delivery of DNA in aqueous solution. The antibody response induced by DNA delivered by gene gun was more cross-reactive than DNA delivered in aqueous solution or after natural infection. This novel approach to vaccination against influenza may afford broader protection against antigenic drift than that provided by natural infection.     

DNA vaccination of pigs with open reading frame 1-7 of PRRS virus

We cloned all open reading frames of a Danish isolate of porcine reproductive and respiratory syndrome (PRRS) virus in DNA vaccination vectors. Pigs were vaccinated using a gene gun with each single construct (ORF1, ORF2, ORF3, ORF4, ORF5, ORF6, or ORF7) or combinations thereof. Vaccination with ORF7 consistently induced antibodies after three vaccinations, while antibodies were only sporadically detected in the remaining groups. After six vaccinations, all pigs were inoculated with PRRS virus and the post-inoculation antibody response was studied. Pigs vaccinated with ORF1 or ORF4 were primed for antibody response against NSP2 or GP4, respectively. Neutralising antibodies were detected in all pigs, with ORF5 vaccinated pigs showing the highest titres.     

Induction of protective immunity by DNA vaccination with Toxoplasma gondii HSP70, HSP30 and SAG1 genes

The vaccination with Toxoplasma gondii heat shock protein 70 (T.g.HSP70) gene (a virulent tachyzoite-specific) induced the most prominent reduction in T. gondii loads in various organs of B6 and BALB/c mice at the acute and chronic phases of toxoplasmosis compared with T.g.HSP30 (a bradyzoite-specific) and SAG1 (a tachyzoite-specific) genes. A single gene gun vaccination with 2 microg of T.g.HSP70 gene induced a significant reduction in the number of T. gondii organisms compared with 50 microg of T.g.HSP70 gene vaccination by intramuscular (i.m.) or intraperitoneal (i.p.) injection. The vaccine effects of T.g.HSP70 gene persisted for more than 3 months.     

Advantage of gene gun-mediated over intramuscular inoculation of plasmid DNA vaccine in reproducible induction of specific immune responses

Utilizing a plasmid DNA encoding a single cytotoxic T lymphocyte (CTL) epitope and that encoding ovalbumin (OVA), we compared the reproducibility in the induction of immune responses by gene gun and intramuscular immunization. As compared to intramuscular inoculation, gene gun DNA immunization appeared to bring about highly reproducible and reliable results in the induction of specific CTL and IFN-gamma production to the CTL epitope and production of anti-OVA IgG. The results obtained by intramuscular inoculation vary significantly. Our data shown here strongly suggest that gene gun immunization of skin is a much more reliable method for DNA vaccination to induce effective immune responses in an animal model.     

Comparison of the CD8+ T cell responses and antitumor effects generated by DNA vaccine administered through gene gun,biojector, and syringe

DNA vaccines have emerged as an attractive approach for antigen-specific cancer immunotherapy. We have previously linked Mycobacterium tuberculosis heat shock protein 70 (HSP70) to human papillomavirus type 16 (HPV-16) E7 in the context of a DNA vaccine. Vaccination with DNA encoding E7/HSP70 has generated a dramatic increase of E7-specific CD8+ T cell precursors and a strong antitumor effect against E7-expressing tumor (TC-1) in vaccinated mice. The success of our strategy has led to two phases I/II clinical trial proposals in patients with HPV-16 associated high-grade squamous intraepithelial lesion (HSIL) of the cervix and in patients with advanced HPV-associated head and neck squamous cell carcinoma (HNSCC). To translate our HPV DNA vaccines into the clinical domain, the efficacy of pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine and of various routes of administrations were assessed in mice. Our results indicated that pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine administered via gene gun generated the highest number of E7-specific CD8+ T cells. In addition, DNA vaccination via gene gun required the least dose to generate similar or slightly better antitumor effects compared to needle intramuscular (i.m.) and biojector administrations. Thus, our data suggest that DNA vaccination via gene gun represents the most potent regimen for DNA administration.     

A glycosylphosphatidylinositol anchor signal sequence enhances the immunogenicity of a DNA vaccine encoding Plasmodium falciparum sexual-stage antigen,Pfs230

Mammalian expression vectors encoding region C of malaria transmission-blocking vaccine candidate Pfs230 (aa 443-1132) with and without a 3' glycosylphosphatidylinositol (GPI) anchor signal sequence were tested for their immunogenicity in mice. The plasmid containing the GPI anchor signal sequence consistently induced higher titers of anti-Pfs230 antibodies using three delivery systems: intramuscular (i.m.), intradermal (i.d.), and gene gun (g.g.). In contrast, the isotype profile elicited varied depending on the delivery system and was not effected by the presence of the GPI anchor sequence. Both gene gun and intradermal administration induced primarily an IgG1 response, while intramuscular injection induced both IgG1 and IgG2a antibodies. Regardless of the mode of delivery, all the plasmids encoding Pfs230 region C primed for a mixed IgG1/IgG2a response to an intraperitoneal (i.p.) injection of E. coli-produced recombinant Pfs230 region C. None of these vaccination strategies were more effective than r230/MBP.C alone in generating malaria transmission-blocking immunity.     

DNA vaccine using hemagglutinating virus of Japan-liposome encapsulating combination encoding mycobacterial heat shock protein 65 and interleukin-12 confers protection against Mycobacterium tuberculosis by T cell activation

We investigated the immunogenicity and protective efficacy of DNA vaccine combinations expressing mycobacterial heat shock protein 65 (Hsp65) and interleukin-12 (IL-12) using gene gun bombardment and the hemagglutinating virus of Japan (HVJ)-liposome method. A mouse IL-12 expression vector (mIL-12 DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. In a mouse model, a single gene gun vaccination with the combination of Hsp65 DNA and mIL-12 DNA provided a remarkably high degree of protection against challenge with virulent Mycobacterium tuberculosis; bacterial numbers were 100-fold lower in the lungs compared to BCG-vaccinated mice. To explore the clinical use of the DNA vaccines, we evaluated HVJ-liposome encapsulated Hsp65 DNA and mIL-12DNA (Hsp65 + mIL-12/HVJ). The HVJ-liposome method improved the protective efficacy of the Hsp65 DNA vaccine compared to gene gun vaccination. Hsp65 + mIL-12/HVJ induced CD8+ cytotoxic T lymphocyte activity against Hsp65 antigen. Most importantly, Hsp65+mIL-12/HVJ vaccination resulted in a greater degree of protection than that evoked by BCG. This protective efficacy was associated with the emergence of IFN-gamma-secreting T cells and activation of proliferative T cells and cytokines (IFN-gamma and IL-2) production upon stimulation with Hsp65 and antigens from M. tuberculosis. These results suggest that Hsp65 + IL-12/HVJ could be a promising candidate for a new tuberculosis DNA vaccine, which is superior to BCG vaccine.     

One-time intradermal DNA vaccination in ear pinnae one year prior to infection protects dogs against rabies virus

Rabid dog exposures result in > 99% of human rabies deaths worldwide. Ninety-eight percent of these cases occur in developing countries. Thus, the best protection against human rabies would be prevention through adequate vaccination of the reservoir population. The difficulty in re-locating ownerless, freely roaming dogs for booster vaccinations, in addition to poor coverage with inadequate vaccines, suggests that a potentially inexpensive vaccine that elicits long-term protection after a single-dose could improve control of canine rabies in developing countries. One solution could be a DNA vaccine. We have previously determined that dogs vaccinated intradermally (i.d.) in ear pinnae with a rabies DNA vaccine expressing a rabies virus glycoprotein (G) produce high levels of neutralizing antibody that persist for at least 6 months. In the present study, we determined whether a one-time i.d. rabies DNA vaccination into ear pinnae 1 year before viral challenge would protect dogs against rabies virus. The dogs did not receive a booster vaccination. All dogs (100%) vaccinated i.d. in each ear pinna with 50 microg of rabies DNA vaccine, or intramuscular (i.m.) with a commercial canine rabies vaccine survived a lethal dose of rabies virus. In contrast, 100% of dogs vaccinated i.m. with 100 microg of rabies DNA developed rabies, as did 100% of negative control dogs that were vaccinated i.d. in each ear pinna or i.m. with DNA that did not express the rabies virus G. The data suggest that a one-time i.d. rabies DNA vaccination into ear pinnae offers a new approach to facilitate control of endemic canine rabies in developing countries.     

Electroporation enhances protective immune response of a DNA vaccine against Japanese encephalitis in mice and pigs

Japanese encephalitis virus (JEV) is a pathogenic cause of Japanese Encephalitis (JE), which is a zoonotic disease transmitted by mosquitoes and amplified by pigs. Infection of JEV may lead to severe neurological sequelae, even death in humans and reproductive disorders in pigs. Vaccination is the only way to control JEV infection in humans. For pigs play important role in the JEV transmission cycle, developing a new veterinary vaccine is considered as a useful strategy for cutting off the transmission route of JEV. We have previously reported that DNA vaccine pCAG-JME, expressing prM-E proteins of JEV, is effective in mice through intramuscular injection (IM). However, the poor immunogenicity, due to low expression of immunogen, is the major obstacle for the development of DNA vaccine in large animals. In the present study, therefore, we immunized mice and pigs with pCAG-JME intramuscularly accompanied with electroporation (EP) stimulation, the attractive gene delivery approach. As compared with IM, EP-mediated vaccination markedly increased the expression of immunogen in the injection site and induced a dose- and time-dependent immune response. 100% survival rate was observed in groups vaccinated with doses ranged from 10 to 100μg, indicating that 10μg of DNA with EP for individual was enough for inducing effective protection in mice. Surprisingly, survival rate and end-point titers of anti-JEV antibodies were higher in mice even at lower dose of DNA (5μg) than that in mice inoculated 100μg through IM. Notably, the prM-E antigens also induced high antibody response in pig, while the neutralizing antibody titer achieved 1:320. Our results suggested that EP-mediated DNA immunization might act as an effective strategy against JEV, at least in pig, and that EP has a potential application prospect in DNA vaccination.     

Gene gun administration of therapeutic HPV DNA vaccination restores the efficacy of prolonged defrosted viral based vaccine

Freshly defrosted vaccines generate promising antitumor immunity by raising both robust CD8 and CD4 responses with a TC1/Th1-dominant cytokine profile. However, prolonged (overnight) defrosted Sindbis virus-E7/HSP70 priming and Vaccinia-E7/HSP70 booster in mouse model only elicited 20% long-term tumor-free survival in comparison with the fresh vaccines. The present study is to search the possible cause of its potency loss, and to evaluate the ability of pcDNA-E7/HSP70 DNA vaccination via gene gun in restoring the efficacy of E7-specific immune responses and antitumor properties. We used prolonged defrosted SINrep5-E7/HSP70 prime and defrostedVac-E7/HSP70 boost subcutaneously, and administered intradermally cluster (3-day interval) gene gun plasmid E7–HSP70DNA vaccine twice, and evaluated its ability to generate antigen-specific cytotoxic CD8+ T-cell responses using flow cytometry as well as antitumor responses using animal positron-emission tomography (PET) imaging. The prolonged defrosted vaccines showed a significant reduction in the infectivity and a significant decrease of CD8+ and CD4+ T-cells immune responses. Administration of cluster gene gun plasmid E7–HSP70DNA twice was also found to lead to restoration of immunity that elicits a full recovery of the antitumor efficacy of the prolonged defrosted vaccines. Our study suggested that adding cluster gene gun plasmid E7–HSP70DNA vaccine twice offered a simple solution in restoring the efficacy of the prime-boost vaccination with viral vectors and has potentially significant clinical applications.     

Chimeric (marker) C-strain viruses induce clinical protection against virulent classical swine fever virus (CSFV) and reduce transmission of CSFV between vaccinated pigs

Two live recombinant vaccines (Flc9 and Flc11) against classical swine fever (CSF) were evaluated for their capacity to reduce transmission of virulent CSF virus (CSFV) among vaccinated pigs. In Flc9 the 5' terminal half of the E2 gene of the C-strain, a CSFV vaccine strain, was exchanged with the homologous gene of the bovine viral diarrhoea virus (BVDV) strain 5250, the E(rns) gene was exchanged likewise in the chimeric Flc11 virus. Both recombinant vaccines induce an antibody response in pigs that can be distinguished from that induced after a wild-type CSFV infection. Four experiments were performed to estimate the reproduction ratio R after different vaccination-challenge intervals. Each group consisted of ten pigs [specified pathogen free (SPF) pigs or conventional pigs] that were vaccinated once, intramuscularly, either with Flc9 or Flc11 virus or that were not vaccinated. Vaccinated and susceptible pigs were challenged intranasally with the virulent CSFV strain Brescia or Behring, 1, 2 or 4 weeks after vaccination. Whether contact-pigs became infected was determined using a CSFV specific E2 (Flc9) or E(rns) (FLc11) antibody ELISA. In the unvaccinated control groups, virus secretion started from day 2 to 4 after inoculation and all contact pigs became infected. Contact pigs became infected in the group of pigs (SPF or conventional) vaccinated once with Flc9 virus and challenged 1-, 2- or 4-weeks later. The estimates of the R in the groups challenged at 1-, 2- and 4-weeks after vaccination were 0.38, 0 and 0.75, respectively. Contact infected pigs were not detected (R=0) in any of the groups of pigs, vaccinated with Flc11, only SPF pigs were used. In order to achieve a statistical significance of R within the vaccinated groups each of the experiments has to be repeated at least once. The R of pigs vaccinated with Flc11 virus and challenged at 1- or 2-weeks after vaccination was however significantly lower that the reproduction ratio of the unvaccinated groups (P=0.013). The R of pigs vaccinated with Flc9 virus and challenged at 1 (conventional pigs) or 2 weeks (SPF pigs) after vaccination was significantly lower that the reproduction ratio of the unvaccinated groups (P=0.013). In conclusion, both chimeric viruses Flc9 and Flc11 provided good clinical protection against a challenge with virulent CSFV at 1 or 2 weeks after vaccination. Further experiments should be carried out to study more aspects of the efficacy of these recombinant viruses before they can be used as a marker vaccine under field circumstances.     

Serological and mucosal immune responses after vaccination and infection with FMDV in pigs

The aim of this study was to determine a possible correlation between humoral immune responses shortly after vaccination and protection against foot-and-mouth disease virus (FMDV) infection and to study the serological and mucosal antibody responses after vaccination and infection. We used three groups of ten pigs, one non-vaccinated group, one group vaccinated with a single dose vaccine and one group vaccinated with a four-fold dose vaccine. At 7 days post vaccination, five pigs per group were challenged intra-dermally with FMDV O TAW 3/97 and the remaining pigs of each group were contact-exposed to the inoculated pigs. In each group, virus excretion and number of contact infections were quantified. The serological and mucosal antibody responses were evaluated until 116 days post infection. Vaccination resulted in a significant decrease of virus excretion. Stepwise linear regression analysis of variables from individual vaccinated pigs revealed the virus excretion after challenge to be correlated with neutralising antibody titres at the day of challenge (p<0.01). In serum and OPF samples comparable isotype-specific antibody responses (IgM, IgG and IgA), could be detected after vaccination as well as after infection. Remarkably, the pigs with the highest IgA responses after vaccination were protected against contact exposure. After infection, a long lasting (up to 116dpi) IgA response was seen in the non-vaccinated and to a lesser extent in the single dose vaccinated pigs. The induction of NSP antibodies in the vaccinated pigs after infection was lower and of shorter duration as compared to the non-vaccinated infected pigs. This experiment shows that vaccination can reduce virus excretion in pigs, which will contribute to reduced transmission of FMDV in the field, even if the pigs are not fully protected. Moreover, vaccines that induce local IgA responses may be more effective, which merits further investigation.