Immunogenicity of tick-borne-encephalitis-virus-(TBEV)-vaccination and impact of age on humoral and cellular TBEV-specific immune responses in patients with rheumatoid arthritis.

Inactivated vaccines, such as tick-borne-encephalitis-virus-(TBEV) vaccine, have been discussed as less immunogenic in elderly and in immunocompromised patients. In this controlled cross-sectional cohort study, the antibody and cellular responses after TBEV-vaccination were investigated in 36 rheumatoid arthritis (RA) patients and 112 healthy controls (HC) by evaluating IgG-anti-TBEV concentration, neutralization and relative avidity index (RAI). Cellular reactivity was assessed by IFNgamma-producing spot-forming-units (SFU) by ELISPOT assay and flow cytometry. RA patients showed lower IgG-anti-TBEV compared to HC, which were influenced by age at and time since last TBEV vaccination and disease duration. High-responders regarding cellular immunity and avidity were less frequent in RA compared to HC. RA patients who had received booster vaccinations were more likely to demonstrate higher IgG-anti-TBEV responses compared to those who had not. In conclusion, RA patients showed a negative effect of age on anti-TBEV-IgG and immunological benefits of timely booster vaccination are suggested.

Omicron (B.1.1.529) BA.1 or BA.2-related effects on immune responses in previously naïve versus imprinted individuals: immune imprinting as an advantage in the humoral immune response against novel variants.

Background: Immune imprinting is a phenomenon in which a person's immune system develops a specific immunological memory of the pathogen or vaccine due to a previous exposure. This memory basically leads to a faster and stronger immune response in a subsequent contact to the same pathogen or vaccine. However, what happens if the pathogen has changed considerably in the meantime due to mutations in the main target region of antibodies, as in the evolution of SARS-CoV-2 from the ancestral strain to B.1.1.529 (Omicron)? In this case, does immune imprinting also confer an advantage in repeated contact and does it lead to a stronger immune response? Methods: To clarify these questions, we investigated the effects of immune imprinting in the context of SARS-CoV-2 by comparing a group of previously infection-naïve versus imprinted study participants and determined differences in humoral and cellular immune responses during and after infection with strain SARS-CoV-2 B.1.1.529 BA.1 and BA.2, respectively. We used a commercial CLIA, immunoblots, IFN-γ ELISpots and a plaque-reduction neutralization test to generate a clear and comparable picture of the humoral and cellular immune response in the two study groups. Results: Imprinted participants developed significantly higher antibody titers and showed significantly stronger neutralization capacity against the ancestral strain, BA.1 and BA.5. The immune response of naïve study participants was narrower and related mainly to the receptor-binding domain, which resulted in a lower neutralization capacity against other strains including BA.5. Naïve study participants showed a significantly higher cellular immune response than the imprinted study group, indicating a higher antigenic challenge. The cellular immune response was directed against general structures of SARS-CoV-2 and not specifically against the receptor-binding domain. Conclusion: Viral variant infection elicits variant-specific antibodies and prior mRNA vaccination or infection with a previous SARS-CoV-2 variant imprints serological responses toward the ancestral strain rather than variant antigens. On the other hand, our study shows that the initially higher specific antibody titers due to former imprinting via vaccination or prior infection significantly increased the humoral immune response, and therefore outperformed the humoral immune response of naïve study participants.

Immune Responses Against SARS-CoV-2 WT and Delta Variant in Elderly BNT162b2 Vaccinees.

Background: Residents of nursing homes are one of the most vulnerable groups during the severe acute syndrome coronavirus 2 (SARS-CoV-2) pandemic. The aim of this study was to characterize cellular and humoral immune responses in >70-year-old participants before vaccination, after first and second vaccination with BNT162b2, in contrast to second-dose-vaccinated participants younger than 60 years. Methods: Peripheral blood mononuclear cells of 45 elderly and 40 younger vaccinees were analyzed by IFNγ ELISpot, specific immunoglobulin G antibody titers against SARS-CoV-2 spike protein, and neutralization abilities against SARS-CoV-2 wild-type (WT) and Delta variant (B.1.617.2). Results: Our results clearly demonstrate a significantly increased T cell response, IgG titers, and neutralization activities against SARS-CoV-2 WT and Delta between first and second vaccination with BNT162b2 in elderly vaccinees, thereby highlighting the importance of the second booster. Interestingly, similar cellular and humoral immune responses against SARS-CoV-2 WT and Delta were found after the second vaccine dose in the young and elderly groups. Conclusions: Our data demonstrate a full picture of cellular and humoral immune responses of BNT162b2-vaccinees in two age cohorts. In all vaccines, SARS-CoV-2 WT-specific antibodies with similar neutralizing activity were detected in all vaccinees. After the second vaccination, neutralization titers against SARS-CoV-2 Delta were impaired in both age groups compared with SARS-CoV-2 WT, thereby emphasizing the need for an additional booster to overcome rising variants of SARS-CoV-2.

Cumulative SARS-CoV-2 mutations and corresponding changes in immunity in an immunocompromised patient indicate viral evolution within the host.

Different scenarios explaining the emergence of novel variants of concern (VOC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported, including their evolution in scarcely monitored populations, in animals as alternative hosts, or in immunocompromised individuals. Here we report SARS-CoV-2 immune escape mutations over a period of seven months in an immunocompromised patient with prolonged viral shedding. Signs of infection, viral shedding and mutation events are periodically analyzed using RT-PCR and next-generation sequencing based on naso-pharyngeal swabs, with the results complemented by immunological diagnostics to determine humoral and T cell immune responses. Throughout the infection course, 17 non-synonymous intra-host mutations are noted, with 15 (88.2%) having been previously described as prominent immune escape mutations (S:E484K, S:D950N, S:P681H, S:N501Y, S:del(9), N:S235F and S:H655Y) in VOCs. The high frequency of these non-synonymous mutations is consistent with multiple events of convergent evolution. Thus, our results suggest that specific mutations in the SARS-CoV-2 genome may represent positions with a fitness advantage, and may serve as targets in future vaccine and therapeutics development for COVID-19.

The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro.

Since its outbreak in 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner. To gain deeper insight into mutation frequency and dynamics, we isolated ten ancestral strains of SARS-CoV-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-qPCR and whole genome sequencing. Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-CoV-2 in vitro. Our results identified a series of adaptive genetic changes, ranging from unique convergent substitutional mutations and hitherto undescribed insertions. The region coding for spike proved to be a mutational hotspot, evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501. We discussed the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-CoV-2 in the adaptation to cell culture. The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-CoV-2 strains. The observed genetic changes of SARS-CoV-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants, along the path of increasing potency of the strain. Competition among a high number of quasi-species in the SARS-CoV-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.

Maintenance of neutralizing antibodies over ten months in convalescent SARS-CoV-2 afflicted patients.

Knowledge of the level and duration of protective immunity against SARS-CoV-2 after primary infection is of crucial importance for preventive approaches. Currently, there is a lack of evidence on the persistence of specific antibodies. We investigated the generation and maintenance of neutralizing antibodies of convalescent SARS-CoV-2-afflicted patients over a ten-month period post-primary infection using an immunofluorescence assay, a commercial chemiluminescent immunoassay and an in-house enzyme-linked neutralization assay. We present the successful application of an improved version of the plaque-reduction neutralization assay which can be analysed optometrically to simplify data interpretation. Based on the results of the enzyme-linked neutralization assay, neutralizing antibodies were maintained in 77.4% of convalescent individuals without relevant decay over ten months. Furthermore, a positive correlation between severity of infection and antibody titre was observed. In conclusion, SARS-CoV-2-afflicted individuals have been proven to be able to develop and maintain neutralizing antibodies over a period of ten months after primary infection. Findings suggest long-lasting presumably protective humoral immune responses after wild-type infection.

Adjunctive homeopathic treatment of hospitalized COVID-19 patients (COVIHOM): A retrospective case series.

BACKGROUND: and purpose. COVID-19 is a novel viral disease causing worldwide pandemia. The aim of this study was to describe the effect of adjunctive individualized homeopathic treatment delivered to hospitalized patients with confirmed symptomatic SARS-CoV-2 infection. PATIENT PRESENTATION: Thirteen patients with COVID-19 were admitted. Mean age was 73.4 ± 15.0 (SD) years. Twelve (92.3%) were speedily discharged without relevant sequelae after 14.4 ± 8.9 days. A single patient admitted in an advanced stage of septic disease died in hospital. A time-dependent improvement of relevant clinical symptoms was observed in the 12 surviving patients. Six (46.2%) were critically ill and treated in the intensive care unit (ICU). Mean stay at the ICU of the 5 surviving patients was 18.8 ± 6.8 days. In six patients (46.2%) gastrointestinal disorders accompanied COVID-19. CONCLUSION: The observations suggest that adjunctive homeopathic treatment may be helpful to treat patients with confirmed COVID-19 even in high - risk patients especially since there is no conventional treatment of COVID-19 available at present.

An in vitro model for assessment of SARS-CoV-2 infectivity by defining the correlation between virus isolation and quantitative PCR value: isolation success of SARS-CoV-2 from oropharyngeal swabs correlates negatively with Cq value.

BACKGROUND: At the beginning of the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), little was known about its actual rate of infectivity and any COVID-19 patient positive in laboratory testing was supposed to be highly infective and a public health risk factor. METHODS: One hundred oropharyngeal samples were obtained during routine work flow of testing symptomatic persons by quantitative polymerase chain reaction (qPCR) and were inoculated onto cell culture of VeroB4 cells to study the degree of infectivity of SARS-CoV-2 in vitro. Quantification by virus titration and an external standard using synthetic RNA gave the breaking point of infectivity in SARS-CoV-2 in vitro. RESULTS: A clear negative correlation (r = - 0.76; p < 0.05) could be asserted between the viral load in quantitative polymerase chain reaction (qPCR) and the probability of a successful isolation in serial isolation experiments of specific oropharyngeal samples positive in qPCR. Quantification by virus titration and an external standard using synthetic RNA indicate a Cq between 27 and 30 in E-gene screening PCR as a breaking point in vitro, where infectivity decreases significantly and isolations become less probable. CONCLUSIONS: This study showed that only the 21% of samples with the highest viral load were infectious enough to transmit the virus in vitro and determined that the dispersion rate in vitro is surprisingly close to those calculated in large retrospective epidemiological studies for SARS-CoV-2. This raises the question of whether this simple in vitro model is suitable to give first insights in dispersion characters of novel or neglected viral pathogens. The statement that SARS-CoV-2 needs at least 40,000 copies to reliably induce infection in vitro is an indication of its transmissibility in Public Health decisions. Applying quantitative PCR systems in diagnosis of SARS-CoV2 can distinguish between patients providing a high risk of transmission and those, where the risk of transmission is probably limited to close and long-lasting contacts.

SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8+ T cell responses.

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.

Home quarantine in COVID-19: A study on 50 consecutive patients in Austria.

A cohort of the first 50 COVID-19 patients in East Tyrol, a region in the southwest of Austria, were monitored in home quarantine. Specific viral ribonucleic acid was detected in throat swabs and stool samples. Analysis indicated a median virus shedding duration of 13 days; however, statistical outliers highlight the importance of consequent testing. This underlines the need of negative throat swabs prior to removing quarantine. We monitored the disease's characteristics via an in-house score called Corona Severity Index, in order to predict an aggravation of the disease. Special attention was paid to early symptoms, such as headache, which appeared to be significantly more common in younger patients (p=0.019). Anosmia and ageusia showed a predominance in female patients (p=0.028). Investigation revealed seven relapses and viral shedding fluctuation in four cases. A follow-up examination shed light on seroconversion which could be observed in 35 of 40 participants. This further clarifies the necessity of establishing discharge standards and follow-up management for COVID-19 patients.

Smell and Taste Disorders in COVID-19 Patients: Objective Testing and Magnetic Resonance Imaging in Five Cases.

Smell and taste disorders are acknowledged as characteristic symptoms for SARS-CoV-2 infection by now. These symptoms have been linked to a neuroinvasive course of disease. In this study, we investigated five consecutive COVID-19 patients with a prolonged course of dysosmia and dysgeusia. Those with objectifiable alteration in taste or smell were subjected to MRI with contrast agent to investigate possible involvement of the central nervous system. We found dysosmia and dysgeusia to be mostly objectifiable, but no evidence for neuroinvasiveness could be detected by MRI in the late stage of the disease. Alterations in taste and smell could be objectified in most patients. Nevertheless, no evidence for a neuroinvasive potential could be identified by MRI, at least in the late stage of disease. We encourage medical professionals to conduct specialized examinations and MRIs in the acute stage of disease, which guarantees an optimum patient care.

Monitoring of alien mosquitoes in Western Austria (Tyrol, Austria, 2018).

Mosquitoes are of major importance to human and animal health due to their ability to transmit various pathogens. In Europe the role of mosquitoes in public health has increased with the introduction of alien Aedes mosquitoes such as the Asian tiger mosquito, Aedes albopictus; the Asian bush mosquito, Ae. japonicus; and Ae. koreicus. In Austria, Ae. japonicus has established populations in various regions of the country. Aedes albopictus is not known to overwinter in Austria, although isolated findings of eggs and adult female mosquitoes have been previously reported, especially in Tyrol. Aedes koreicus had not so far been found in Austria. Within the framework of an alien mosquito surveillance program in the Austrian province of Tyrol, ovitraps were set up weekly from May to October, 2018, at 67 sites- 17 in East Tyrol and 50 in North Tyrol. Sampling was performed at highways and at urban and rural areas. DNA obtained from mosquito eggs was barcoded using molecular techniques and sequences were analysed to species level. Eggs of alien Aedes species were found at 18 out of 67 sites (27%). Both Ae. albopictus and Ae. japonicus were documented at highways and urban areas in both East and North Tyrol. Aedes koreicus was found in East Tyrol. During this mosquito surveillance program, eggs of Ae. albopictus, Ae. japonicus, and Ae. koreicus were documented in the Austrian province of Tyrol. These findings not only show highways to be points of entry, but also point to possible establishment processes of Ae. japonicus in Tyrol. Moreover, Ae. koreicus was documented in Austria for the first time.

Monitoring of alien mosquitoes of the genus Aedes (Diptera: Culicidae) in Austria.

Systematic, continuous mosquito surveillance is considered the most reliable tool to predict the spread and establishment of alien mosquito species such as the Asian tiger mosquito (Aedes albopictus), Japanese bush mosquito (Aedes japonicus), and the transmission risk of mosquito-borne arboviruses to humans. Only single individuals of Ae. albopictus have been found in Austria so far. However, it is likely that the species will be able to establish populations in the future due to global trade and traffic as well as increasing temperatures in the course of global climate change. In summer 2017, a project surveilling the oviposition of newly introduced Aedes mosquitoes, using ovitraps, was set up by means of citizen scientists and researchers and was performed in six federal provinces of Austria-Tyrol, Carinthia, Vienna, Lower Austria, Styria, and Burgenland. Eggs of Ae. albopictus were identified in Tyrol during the months August and September, while Ae. japonicus was found in Lower Austria, Styria, and Burgenland. In Vienna and Carinthia, all ovitraps were negative for Aedes eggs; however, Ae. japonicus was found for the first time in Vienna in July 2017 during routine sampling of adult mosquitoes. With this project, we demonstrated the benefits of citizen scientists for ovitrap-based mosquito surveillance. The finding of Ae. albopictus eggs in Northern Tyrol is not yet a proof of the establishment of a self-sustaining population, although it indicates the ongoing introduction of this species along main traffic routes from Italy, where this mosquito is well established. The risk of establishment of the tiger mosquito in the Lower Inn Valley is therefore a given and informing the public about preventive measures to hinder and delay this development is highly recommended.

Practical Guidelines for Studies on Sandfly-Borne Phleboviruses: Part II: Important Points to Consider for Fieldwork and Subsequent Virological Screening.

In this series of review articles entitled "Practical guidelines for studies on sandfly-borne phleboviruses," the important points to be considered at the prefieldwork stage were addressed in part I, including parameters to be taken into account to define the geographic area for sand fly trapping and how to organize field collections. Here in part II, the following points have been addressed: (1) factors influencing the efficacy of trapping and the different types of traps with their respective advantages and drawbacks, (2) how to process the trapped sand flies in the field, and (3) how to process the sand flies in the virology laboratory. These chapters provide the necessary information for adopting the most appropriate procedures depending on the requirements of the study. In addition, practical information gathered through years of experience of translational projects is included to help newcomers to fieldwork studies.

Guidelines for the Detection of Babesia and Theileria Parasites.

The genera Babesia and Theileria (phylum Apicomplexa, order Piroplasmida) are mainly transmitted by Ixodid ticks in which the sexual part of their life cycle followed by sporogony takes place. They include protozoan parasites that infect erythrocytes of a variety of vertebrate hosts, including domestic and wild animals, with some Babesia spp. also infecting humans. Babesia sporozoites transmitted in the tick's saliva during the bloodmeal directly infect erythrocytes, where they asexually multiply to produce pear-shaped merozoites in the process of merogony; whereas a pre-erythrocytic schizogonic life stage in leukocytes is found in Theileria and precedes merogony in the erythrocytes. The wide spectrum of Babesia and Theileria species and their dissimilar characteristics with relation to disease severity, transmission, epidemiology, and drug susceptibility stress the importance of accurate detection of babesiosis and theileriosis and their causative agents. These guidelines review the main methods currently used for the detection of Babesia and Theileria spp. for diagnostic purposes as well as epidemiological studies involving their vertebrate hosts and arthropod vectors. Serological methods were not included once they did not indicate current infection but rather exposure.

Practical Guidelines for Studies on Sandfly-Borne Phleboviruses: Part I: Important Points to Consider Ante Field Work.

The purpose of this review is to provide practical information to help researchers intending to perform "from field to laboratory" studies on phleboviruses transmitted by sandflies. This guideline addresses the different steps to be considered starting from the field collection of sandflies to the laboratory techniques aiming at the detection, isolation, and characterization of sandfly-borne phleboviruses. In this guideline article, we address the impact of various types of data for an optimal organization of the field work intending to collect wildlife sandflies for subsequent virology studies. Analysis of different data sets should result in the geographic positioning of the trapping stations. The overall planning, the equipment and tools needed, the manpower to be deployed, and the logistics to be anticipated and set up should be organized according to the objectives of the field study for optimal efficiency.

Guidelines for the Direct Detection of Anaplasma spp. in Diagnosis and Epidemiological Studies.

The genus Anaplasma (Rickettsiales: Anaplasmataceae) comprises obligate intracellular Gram-negative bacteria that are mainly transmitted by ticks, and currently includes six species: Anaplasma bovis, Anaplasma centrale, Anaplasma marginale, Anaplasma phagocytophilum, Anaplasma platys, and Anaplasma ovis. These have long been known as etiological agents of veterinary diseases that affect domestic and wild animals worldwide. A zoonotic role has been recognized for A. phagocytophilum, but other species can also be pathogenic for humans. Anaplasma infections are usually challenging to diagnose, clinically presenting with nonspecific symptoms that vary greatly depending on the agent involved, the affected host, and other factors such as immune status and coinfections. The substantial economic impact associated with livestock infection and the growing number of human cases along with the risk of transfusion-transmitted infections, determines the need for accurate laboratory tests. Because hosts are usually seronegative in the initial phase of infection and serological cross-reactions with several Anaplasma species are observed after seroconversion, direct tests are the best approach for both case definition and epidemiological studies. Blood samples are routinely used for Anaplasma spp. screening, but in persistently infected animals with intermittent or low-level bacteremia, other tissues might be useful. These guidelines have been developed as a direct outcome of the COST action TD1303 EURNEGVEC ("European Network of Neglected Vectors and Vector-Borne Diseases"). They review the direct laboratory tests (microscopy, nucleic acid-based detection and in vitro isolation) currently used for Anaplasma detection in ticks and vertebrates and their application.

Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay.

The obligate intracellular bacterium Chlamydia abortus is the causative agent of enzootic abortion of ewes and poses a significant zoonotic risk for pregnant women. Using proteomic analysis and gene expression library screening in a previous project, we identified potential virulence factors and candidates for serodiagnosis, of which nine were scrutinized here with a strip immunoassay. We have shown that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, Pmp13G) and virulence-associated (CPAF, TARP, SINC) antigens. While the latter disappeared within 18 weeks following abortion in a majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, nonaborting experimentally infected sheep developed mainly antibodies to surface antigens (MOMP, MIP, Pmp13G), all of which did not persist. We were also able to detect antibodies to these surface antigens in C abortus-infected women who had undergone septic abortion, whereas a group of shepherds and veterinarians with occupational exposure to C abortus-infected sheep revealed only sporadic immune responses to the antigens selected. The most specific antigen for the serodiagnosis of human C abortus infections was Pmp13G, which showed no cross-reactivity with other chlamydiae infecting humans. We suggest that Pmp13G-based serodiagnosis accomplished by the detection of antibodies to virulence-associated antigens such as CPAF, TARP, and SINC may improve the laboratory diagnosis of human and animal C abortus infections.

Borrelia burgdorferi sensu stricto and Borrelia afzelii: Population structure and differential pathogenicity.

MultiLocus sequence typing (MLST) is considered a powerful method to unveil relationships within bacterial populations and it constitutes an economical and fast alternative to whole genome sequencing. We used this method to understand whether there are differences in human pathogenicity within and between different Borrelia burgdorferi sensu lato species. Therefore, 136 strains from human patients or ticks from Europe were included in MLST analyses. The scheme employed used eight chromosomally located housekeeping genes (i.e. clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA). We investigated Borrelia afzelii, one of the predominant species in Europe, and B. burgdorferi sensu stricto (s.s.), because it allowed comparative analysis to strains from the USA. We typed 113 patient isolates as well as 23 tick isolates. For further comparative purposes an additional 746 strains from Europe and the USA were included from the MLST website http://borrelia.mlst.net. We observed an overlap of the B. burgdorferi s.s. populations from Europe and the USA isolated from human patients while there was no overlap of the populations found in tick vectors. Further results indicate that B. afzelii was significantly less associated with disseminated infection than B. burgdorferi s.s. and that B. burgdorferi s.s. from Europe caused neuroborreliosis to a significantly greater extent than B. afzelii or B. burgdorferi s.s. in the USA. Our data suggest that there may be an evolutionary basis of differential interspecies pathogenicity in Borrelia. This was not evident within Borrelia species: we found the same sequence types in patients with disseminated or localized symptoms when the number of strains was sufficiently high. We hypothesize that the finding that B. burgdorferi s.s. in Europe is much more associated with neuroborreliosis than in the USA maybe linked to factor(s) related to the human host, the tick vector or the bacterium itself (e.g. plasmid content and structure).